Research ArticleThe Hair Growth-Promoting Effect ofRumex japonicus Houtt Extract

Hyunkyoung Lee12 Na-Hyun Kim3 Hyeryeon Yang1 Seong Kyeong Bae14

Yunwi Heo1 Indu Choudhary1 Young Chul Kwon1 Jae Kuk Byun1 Hyeong Jun Yim1

Byung Seung Noh5 Jeong-Doo Heo3 Euikyung Kim12 and Changkeun Kang12

1College of Veterinary Medicine Gyeongsang National University Jinju 52828 Republic of Korea2Institute of Animal Medicine Gyeongsang National University Jinju 52828 Republic of Korea3Gyeongnam Department of Environment Toxicology and Chemistry Toxicity Screening Research CenterKorea Institute of Toxicology Jinju 52834 Republic of Korea4Institutes of Agriculture and Life Science Gyeongsang National University Jinju 52828 Republic of Korea5Biobeauty Korea Co Ltd Kyungsung University Business Incubator Busan 48434 Republic of Korea

Correspondence should be addressed to Changkeun Kang ckkanggnuackr

Received 5 August 2016 Revised 4 October 2016 Accepted 25 October 2016

Academic Editor Yoshiyuki Kimura

Copyright copy 2016 Hyunkyoung Lee et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Rumex japonicusHoutt is traditionally used as amedicinal plant to treat patients suffering from skin disease in Korea However thebeneficial effect of Rumex japonicusHoutt on hair growth has not been thoroughly examinedTherefore the present study aims toinvestigate the hair growth-promoting effect of Rumex japonicus (RJ) Houtt root extract using human dermal papilla cells (DPCs)HaCaT cells and C57BL6 mice model RJ induced antiapoptotic and proliferative effects on DPCs and HaCaT cells by increasingBcl-2Bax ratio and activating cellular proliferation-related proteins ERK and Akt RJ also increased 120573-catenin via the inhibition ofGSK-3120573 In C57BL6 mice model RJ promoted the anagen induction and maintained its period Immunohistochemistry analysisdemonstrated that RJ upregulated Ki-67 and 120573-catenin expressions suggesting that the hair growth effect of RJ may be mediatedthrough the reinforcement of hair cell proliferationThese results provided important insights for the possible mechanism of actionof RJ and its potential as therapeutic agent to promote hair growth

1 Introduction

Hair follicle is an organ composed with epithelial and mes-enchymal tissues It continuously cycles through a growthphase (anagen) an involution phase (catagen) and a restingphase (telogen) [1] Hair loss is recently highlighted as anincreasing disease characterized by the abnormal hair cyclesand unstable hair follicle maintenance Hormone imbalancestress nutritional deficiency aging and seborrheic dermatitiscan cause hair loss [2ndash5] Although hair loss is not lifethreatening it causes serious problems (social interactionand psychology) for numerous people in the world To dateFood and Drug Administration approved drugs in USAfor the treatment of hair loss are only two finasteride and

minoxidil Finasteride is originally developed as a treatmentof benign prostatic hypertrophy but is found to have pro-moting effect on hair growth It is a 5120572-reductase inhibitorthat prevents conversion testosterone to dihydrotestosterone(DHT) resulting in a decrease of androgen activity in thescalp [6] Minoxidil is an antihypertensive vasodilator agentIt is also known to be effective in androgenic alopeciaHowever the efficacy disappears within a few months afterstopping therapy and side effects are reported to cause allergicdermatitis and mild scalp irritation Use of two drugs islimited by low efficacy and unfavorable effect [7] Thereforethe interest and importance to develop novel preventiveandor therapeutic materials for hair loss have increasedrecently

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 1873746 10 pageshttpdxdoiorg10115520161873746

2 Evidence-Based Complementary and Alternative Medicine

The promotion of hair follicle growth is strongly influ-enced by various hormones growth factors and develop-ment-related proteins Among themWnt120573-catenin pathwayplays an important role in the hair growth cycle 120573-catenin inDPC is markedly activated during anagen phase resulting ininitiation of hair follicle formation stimulating hair growthand differentiation [8ndash10] In 120573-catenin knock-out mice thecatagen and telogen phases induce premature condition andregeneration of hair follicle is the failure [11] In the absence ofWnt 120573-catenin is ordinarily phosphorylated in the cytoplasmby glycogen synthase GSK-3b (beta) and kinase-3b tagged byan ubiquitin and then dissolved by the proteasome HoweverWnt protein binds to receptors of Frizzled family and 120573-catenin is accumulated in the cytoplasm and translocatedinto the nucleus resulting in induction of growth signalsfor hair follicle progenitors and promoting differentiationof hair follicular keratinocyte [9 12] Therefore GSK-3 isa key regulator in 120573-catenin pathway Targeting the 120573-catenin pathway is an ideal clinical therapy for preventinghair loss and enhancing hair regrowth Therefore manyresearchers have reported chemicals and natural extracts thatstimulate hair growth by targeting the 120573-catenin pathway[13ndash15]

Rumex japonicusHoutt a perennial herb plant belongingto the family Polygonaceae is widely distributed in theEast Asia (China Japan and Korea) It has been used fortreating heat phlegm jaundice constipation uterine hemor-rhage and skin disease in traditional medicine [16ndash20] RjaponicusHoutt contains a large number of anthraquinonesoxanthrones and flavones and several reports demonstrateantibacterial anti-inflammatory and antioxidant effects inskin disease and inhibitory effect of atopic dermatitis [1618ndash23] However the hair growth-promoting effect of Rjaponicus Houtt has not been scientifically proven yet Inthis study we investigated for the first time the stimulatingeffect of dried roots of R japonicusHoutt extract (RJ) and itsmechanismonhair growth For this we investigated the effectof RJ on cultured DPCs HaCaT cells and C57BL6 mice

2 Materials and Methods

21 Materials Bovine serum albumin (BSA) DulbeccorsquosModified Eaglersquos Medium (DMEM) Fetal Bovine Serum(FBS) penicillin streptomycin and trypsin were purchasedfrom Gibco-BRL (Grand Island NY USA) Dimethyl sulfox-ide (DMSO) and 3-(45-dimethylthiazol-2-yl)-25 diphenyl-tetrazolium bromide (MTT) were from Sigma-Aldrich Inc(St Louis MO USA) Antibodies for Bcl-2 Bax phospho-Akt phospho-ERK12 120573-catenin phospho-GSK3120573 and 120573-actin were obtained fromCell Signaling Technology (BeverlyMA USA)Minoxidil was purchased fromHyundai Pharma-ceuticals (Seoul Korea) All other reagents used were of thepurest grade available

22 Preparation of Dried Roots of R japonicus Houtt ExtractThe dried roots of R japonicus Houtt were provided byBiobeauty Korea Co Ltd (Busan Korea) The roots werewashed 3 times with tapwater to remove impurities and driedat room temperature and then stored at 4∘C until use Dried

roots were ground into powder with a grinder and extracted3 times with 95 ethanol at 25∘C for 3 days after which theextract was filtered through the Advantech number 3 filterpaper (Osaka Japan)Thefiltered liquidwas evaporated usingrotary vacuum evaporator (Tokyo Rikakikai Co Ltd TokyoJapan) The final step was lyophilization under vacuum todryness

23 Cell Culture Human dermal papilla cells (DPCs) wereobtained from Prof Ohsang Kown in the Department ofDermatology College of Medicine at Seoul National Uni-versity Human keratinocyte (HaCaT) cells were purchasedfrom American Type Culture Collection (ATCC) DPC isobtained from occipital scalp tissue containing more than100 hair follicles in twelve healthy male volunteers (20sim50ages Korean) according to the approval of Seoul NationalUniversity Hospital Institutional Review Board (IRB numberH-1112-096-390) Before starting this experiment the DPCwas cultured for three passages They were maintainedin DMEM supplemented with 10 FBS and 100120583gmLpenicillin-streptomycin-amphotericin B solution They weregrown asmonolayer cultures and kept at 37∘C in a humidifiedatmosphere with 5 CO

2for growth

24 MTT Assay Cell viability was measured by the MTTassay as previously described [24] Briefly the cells wereplated at a density of 4 times 104 cellswell in 24-well plates andcultured overnight in growth DMEM media After changinglow serum media (1 FBS) the cells were treated withvarious concentrations of RJ and incubated for 1 and 3 daysMTT solution (5mgmL) was then added to each well andincubated for additional 3 h at 37∘C Finally DMSO wasadded to solubilize the formazan salt and the amount offormazan generated was determined by measuring opticaldensity (OD) at 540 nm using GENios microplate spec-trophotometer (PowerWaveXS BioTek Instruments IncWinooski USA)

25 Western Blot Analysis Cells plated on microplate disheswere incubated with various concentrations of RJ and thenwashed with cold PBS The treated cells were collected byscraping with 300 120583L of RIPA buffer (iNtRON biotechnologySeongnam Korea) containing protease inhibitor cocktail(Thermo Fischer Scientific Pierce IL USA)The scraped cellswere allowed to lyse for additional 30min on ice with peri-odic vortexing Cell debris was removed by centrifugation(13000timesg at 4∘C for 30min) and the resulting supernatantwas collected and determined for its protein concentrationusing a Bio-Rad protein assay reagent (Bio-Rad CA USA)The samples were separated on 10 SDS-polyacrylamidegel and then transferred to PVDF membranes (Bio-RadCA USA) Western blot was probed with specific primaryantibodies overnight at 4∘C After washing the membraneswere incubated with horseradish peroxidase-conjugated sec-ondary antibody (Bethyl Laboratories Inc MontgomeryUSA) for 1 hr at room temperature The blots were visualizedby using an enhanced chemiluminescence method (ECLAmershamBiosciences Buckinghamshire UK) and analyzedusing ChemiDoc XRS (Bio-Rad CA USA) Densitometry

Evidence-Based Complementary and Alternative Medicine 3

analysis was performed with a Hewlett-Packard scanner andNIH Image software (Image J)

26 Immunofluorescence Analysis TheDPC and HaCaT cellswere grown on cell culture slide (SPL Life Science PocheonKorea) to 40ndash50 confluence After incubation for 24 h thecells were treated with RJ for another 24 h and then washedwith PBS three times for 5min The cells were fixed with 4paraformaldehyde for 10min and blocked with 3 FBS for1 h at room temperature (RT) Primary antibody (120573-catenin)was added to the cells and incubated overnight at 4∘C Thefollowingmorning the slides were washed three times in PBSand incubated with goat anti-rabbit immunoglobulin conju-gated with red-fluorescent dye for 1 h at RT Subsequentlythe stained cells were dropped with Vectashield mountingmedium containing DAPI to counterstain cell nuclei (VectorLaboratories CA USA) The samples were observed with aLeica fluorescence microscope (Leica Wetzlar Germany)

27 Experimental Study with RJ for Hair Growth This studywas performed by the Korea Institute of Toxicology (JinjuKorea) in accordance with the Institutional Animal Care andUse Committee (IACUC 1504-0003) 6-week-old C57BL6mice were purchased from Samtako Inc (Osan Korea) andadapted for 1weekThedorsal portion of 7-week-oldmicewasshaved with an animal clipper at which mouse hair follicleswere synchronized in the telogen stage Forty animals in 4randomized groups (119899 = 10) were used for studying thehair growing activity of RJ RJ was dissolved in a vehiclemixture (methanol PBS 60 40 vv)The animals in group1 were topically treated with vehicle mixture animals ingroups 2 and 3 were treated with RJ 4 and 8mgmL on dorsalskin once a day for 25 days Animals in group 4 (positivecontrol group) were treated with 5 minoxidil in the sameschedule with RJ treatment Animals were kept in isolationfor a certain amount of time and then housed back to separatecages To assess changes in hair morphology the mice wereanesthetized and photographed at 0 6 11 16 21 and 26days The ratio of hair growth (grown areashaved area) onthe photographs was analyzed using image analysis programNIS-Elements Basic Research Software (Nikon InstrumentsINC Tokyo Japan)

28 Histological Examination of Hair Follicles After 26 daysthemicewere sacrificed and their shaved skinswere collectedThe dorsal skin was cut into two parts for hematoxylinand eosin (HampE) staining and immunohistochemistry (IHC)uses The skin was fixed with 10 neutral formalin andembedded in paraffin blocks to obtain longitudinal andtransverse section 5120583m tissue sections were stained withhematoxylin and eosin (HampE) to confirm cycle of hair follicleThe percentage of hair follicles in each defined cycle stage atday 26 was calculated The other part of the tissue was frozenin liquid nitrogen for immunohistochemistry

29 Immunohistochemistry IHC for Ki-67 and120573-cateninwasperformed using rabbit monoclonal antibodies Immunore-activity was visualized with an avidin-biotin peroxidasereaction (PK-4001 Vectastain ABC kit) The peroxidase

reaction was developed using a 3 31015840-diaminobenzidinetetrahydrochloride (D-5905 Sigma)The sections were coun-terstained with hematoxylin before being mounted

210 Hair Morphological Change Using Scanning Electron Mi-croscope (SEM) The samples were placed on an adhesivecarbon tape attached to a metal stub and coated withplatinumusing a sputtering device Photomicrographs of hairfiber were obtained by a SEMusing JSM-7610F (JEOL TokyoJapan)

211 Statistical Analysis The results are expressed as meanplusmn standard deviation (SD) One-way analysis of variance(ANOVA) was used to evaluate the significance of differencebetween two mean values 119901 lt 001 was considered to bestatistically significant

3 Results

31 RJ Promotes DPC and HaCaT Cells Proliferation DPCand HaCaT cells were used as models in this study becausethey are important in the growth of hair To determinethe effects of RJ on DPC and HaCaT cells MTT assaywas performed In addition 100 120583M minoxidil (Mi) wasused as a positive control RJ enhanced the proliferation ofDPCs by 1321 and 1479 at the concentrations of 50 and100 120583gmL for 3 days compared with the control (Figure 1(a))In DPC treatment of RJ 100 120583gmL for 3 days exhibited thegreatest effectiveness which is stronger thanMi RJ treatmentsignificantly also promoted the proliferation of HaCaT cellsby 1313 at 50120583gmL for 3 days compared with the control(Figure 1(b)) The results suggest that RJ might have hairgrowth-promoting effect via the proliferation of DPC andHaCaT cells

32 RJ Activates Akt ERK12 andWnt120573-Catenin in DPC andHaCaT Cells Minoxidil has been well known to enhanceproliferation of hair cells through MAPK kinase family(ERK12 JNK12 and p38) and Akt signaling pathwaysTherefore the mechanism of proliferative effects by RJ inDPC and HaCaT cells was determined by western blot Asshown in Figure 2(a) the expression level of p-Akt wassteadily increased for up to 24 h in time-dependent manneron both DPC and HaCaT cells while p-ERK12 level waselevated up to 6 h in DPC and 1 h in HaCaT cells by treatmentof RJ 50120583gmL The phosphorylation of JNK12 and p38did not change in DPC and HaCaT cells (data not shown)Additionally RJ increased the expressions of 120573-catenin andp-GSK3120573 for 12 h in both DPC and HaCaT cells Treatmentwith dose-dependent manner of RJ increased the p-Aktand p-ERK12 as well as 120573-catenin and p-GSK3120573 in DPCand HaCaT cells for 24 h (Figure 2(b)) Nuclear 120573-cateninexpression also was increased by RJ treatment in both cells(Figure 2(c)) Interestingly the expression levels of 120573-cateninand p-GSK3120573 were greatly elevated by treatment with RJ butnot in Mi Moreover we confirmed significant activation of120573-catenin in DPC and HaCaT cells treated with RJ 50 120583gmLby immunofluorescence In control cells 120573-catenin was veryweakly detected in both cells However its expression was

4 Evidence-Based Complementary and Alternative Medicine

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Figure 1 Effect of RJ on proliferation in DPC and HaCaT cells (a) DPC and (b) HaCaT cells were treated with RJ (5 10 50 100 and500 120583gmL) for 1 and 3 days Cell viability was then determined in a MTT assay Minoxidil (Mi 100120583M) was used as positive control Thedata shown are the mean plusmn SD of three independent experiments Significant difference from control group lowast119901 lt 001

increased and detected in cytoplasm by treatment of RJ(Figures 2(d) and 2(e)) Similarly with western blotting resultexpression of 120573-catenin was increased by RJ in immunofluo-rescence data These results suggest that RJ can increase theproliferation of DPC and HaCaT cells via ERK12 Akt and120573-catenin signaling pathways

33 Regulation of RJ on Apoptosis-Related Proteins Since RJinduced the growth-promoting effect of DPC and HaCaTcells we examined the expression levels of the anti- andproapoptotic protein in the two Bcl-2 family proteins Inresponse to treatment of DPC with RJ the level of antiapop-totic Bcl-2 protein dose-dependently increased by 12- to 2-fold and the level of proapoptotic Bax protein significantlydecreased at only 50 120583gmL (Figure 3(a)) Additionally inHaCaT cells RJ induced significant upregulation of Bcl-2 pro-tein and downregulation of Bax protein in dose-dependentmanner (Figure 3(b)) Bcl-2Bax ratio was increased by 11- to24-fold and 12- to 22-fold in DPC and HaCaT cells as doseincreased from 125 to 50 120583gmL (Figures 3(a) and 3(b))

34 RJ Promotes Induction of Anagenic Phase in TelogenicC57BL6 Mice The black pigmentation was taken as evi-dence for transition of hair follicles from telogen to anagenphase To evaluate the hair growth-promoting activity of RJwe topically applied daily RJ onto the shaved back of telogenicC57BL6 mice for 25 days 5 Mi was separately applied asa positive control We evaluated the degree of hair growthby observing the skin color every 5 days At 11 days RJ-treated group showed partly black coloration and short hair

in the shaved skin while less visible black pigmentation wasobserved in vehicle group (Figure 4(a)) At 16 and 21 days RJ-treated group(8mgmL) showed markedly hair growth overhalf of mice At 26 days in RJ-treated group (8mgmL) themice backwas coveredwith overall hair except for threemicewhereas the vehicle group showed relatively less hair growthIn the Mi-treated group the change in the color of back skinwas earlier than in the other groups and covered completelyafter 26 days except for two mice The effect on hair growthof RJ treatment was assessed by measuring regrown area andshaved area Mi-treated group showed 839 of hair growthscore at 26 days compared with the vehicle group which keptshowing 26 of hair growth score Although less effectivethan Mi-treated group RJ-treated group exerted 399 and545 of hair growth score at 4 and 8mgmL at 26 days(Figure 4(b)) These results indicate that RJ promoted hairgrowth and telogen to anagen transition in C57BL6 mice

35 Effects of RJ on Development of Hair Follicle An increasein the number and the size of hair follicles during anageninduction is reported [25] To investigate whether RJ inducesthe anagen induction in hair follicle HampE staining wasperformed In the representative longitudinal sections thelength of hair follicle in RJ-treated group was longer andthe size was larger than in the vehicle group (Figure 5(a))Skin thickness also was increased in RJ- and Mi-treatedgroups compared to vehicle group Additionally as a result ofanalyzing hair cycle RJ-treated groups showed that anagenphase of hair follicle was increased over 2-fold and telogenphase was decreased by 6-fold as compared with vehicle

Evidence-Based Complementary and Alternative Medicine 5

DPCs0 1 3 6 12 24 (h)

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Figure 2 Effect of RJ on ERK12 Akt p-GSK3120573 and120573-catenin expressions inDPC andHaCaT cells (a) Time-dependent effects of p-ERK12p-Akt p-GSK3120573 and 120573-catenin expressions were determined in cells treated with RJ (50120583gmL) for the indicated times (b) Dose-dependenteffects of p-ERK12 p-Akt p-GSK3120573 and 120573-catenin were detected in RJ-treated cells using western blotting (c) To confirm translocation of120573-catenin into nucleus nuclear 120573-catenin expression was determined by western blotting (d and e)The 120573-catenin expression was determinedby immunofluorescence staining with anti-120573-catenin in DPC and HaCaT cells

Bcl-2

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(a)

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Figure 3 Effects of RJ on expressions of Bcl-2 and Bax proteins (a) DPC and (b) HaCaT cells were treated with RJ (0 125 25 and 50120583gmL)The levels of Bcl-2 and Bax were analyzed by western blotting using specific monoclonal antibodies 120573-Actin was used as a loading controlThe data shown are the mean plusmn SD of six independent experiments Significant difference from control group lowast119901 lt 001

6 Evidence-Based Complementary and Alternative MedicineVe

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Figure 4 Effect of RJ on the anagen induction in C57BL6 mice (a) After shaving the back skin was treated with vehicle RJ and 5 Mievery day for 25 daysThe back skin was photographed at 0 6 11 16 21 and 26 days after shaving (b) Morphological change in C57BL6 micetreated with vehicle RJ and Mi was analyzed Parts of the shaved and the hair growth area measured using the NIS-Elements Basic ResearchSoftware The data shown are the mean plusmn SD (119899 = 10) Significant difference from vehicle group lowast119901 lt 001

group The percentage ratio of anagen to telogen phase ofhair growth inRJ-treated group (8mgmL)was also increasedfrom 26 58 to 66 12 in comparison with vehicle group(Figure 5(b)) These results suggest that RJ enhanced the hairgrowth which appeared in the increase in the hair folliclenumber size length and skin thickness and induction ofearly telogen to anagen conversion compared to the vehiclegroup

36 Involvement between the Promoting Hair Growth Effectand 120573-Catenin To confirm the proliferation and promotingeffect in hair cells we performed the IHCanalysis usingKi-67which is cellular proliferationmarker [26] Immunoreactivity

to Ki-67 revealed that RJ 8mgmL and Mi enhanced morecellular proliferation within matrix part of hair follicles thanvehicle group (Figure 6) To understand the mechanism ofinducing anagen phase in RJ-treated group the expression of120573-catenin was confirmed using IHC analysis 120573-catenin pro-tein expression levels were strongly detected in RJ 8mgmLcompared to vehicle group (Figure 6)

4 Discussion

Hair loss is generally not a life-threatening disorder but it ispsychological disease that has an impact on social interactionTo date there are only two antihair loss drugs finasteride and

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

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Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

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Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

The promotion of hair follicle growth is strongly influ-enced by various hormones growth factors and develop-ment-related proteins Among themWnt120573-catenin pathwayplays an important role in the hair growth cycle 120573-catenin inDPC is markedly activated during anagen phase resulting ininitiation of hair follicle formation stimulating hair growthand differentiation [8ndash10] In 120573-catenin knock-out mice thecatagen and telogen phases induce premature condition andregeneration of hair follicle is the failure [11] In the absence ofWnt 120573-catenin is ordinarily phosphorylated in the cytoplasmby glycogen synthase GSK-3b (beta) and kinase-3b tagged byan ubiquitin and then dissolved by the proteasome HoweverWnt protein binds to receptors of Frizzled family and 120573-catenin is accumulated in the cytoplasm and translocatedinto the nucleus resulting in induction of growth signalsfor hair follicle progenitors and promoting differentiationof hair follicular keratinocyte [9 12] Therefore GSK-3 isa key regulator in 120573-catenin pathway Targeting the 120573-catenin pathway is an ideal clinical therapy for preventinghair loss and enhancing hair regrowth Therefore manyresearchers have reported chemicals and natural extracts thatstimulate hair growth by targeting the 120573-catenin pathway[13ndash15]

Rumex japonicusHoutt a perennial herb plant belongingto the family Polygonaceae is widely distributed in theEast Asia (China Japan and Korea) It has been used fortreating heat phlegm jaundice constipation uterine hemor-rhage and skin disease in traditional medicine [16ndash20] RjaponicusHoutt contains a large number of anthraquinonesoxanthrones and flavones and several reports demonstrateantibacterial anti-inflammatory and antioxidant effects inskin disease and inhibitory effect of atopic dermatitis [1618ndash23] However the hair growth-promoting effect of Rjaponicus Houtt has not been scientifically proven yet Inthis study we investigated for the first time the stimulatingeffect of dried roots of R japonicusHoutt extract (RJ) and itsmechanismonhair growth For this we investigated the effectof RJ on cultured DPCs HaCaT cells and C57BL6 mice

2 Materials and Methods

21 Materials Bovine serum albumin (BSA) DulbeccorsquosModified Eaglersquos Medium (DMEM) Fetal Bovine Serum(FBS) penicillin streptomycin and trypsin were purchasedfrom Gibco-BRL (Grand Island NY USA) Dimethyl sulfox-ide (DMSO) and 3-(45-dimethylthiazol-2-yl)-25 diphenyl-tetrazolium bromide (MTT) were from Sigma-Aldrich Inc(St Louis MO USA) Antibodies for Bcl-2 Bax phospho-Akt phospho-ERK12 120573-catenin phospho-GSK3120573 and 120573-actin were obtained fromCell Signaling Technology (BeverlyMA USA)Minoxidil was purchased fromHyundai Pharma-ceuticals (Seoul Korea) All other reagents used were of thepurest grade available

22 Preparation of Dried Roots of R japonicus Houtt ExtractThe dried roots of R japonicus Houtt were provided byBiobeauty Korea Co Ltd (Busan Korea) The roots werewashed 3 times with tapwater to remove impurities and driedat room temperature and then stored at 4∘C until use Dried

roots were ground into powder with a grinder and extracted3 times with 95 ethanol at 25∘C for 3 days after which theextract was filtered through the Advantech number 3 filterpaper (Osaka Japan)Thefiltered liquidwas evaporated usingrotary vacuum evaporator (Tokyo Rikakikai Co Ltd TokyoJapan) The final step was lyophilization under vacuum todryness

23 Cell Culture Human dermal papilla cells (DPCs) wereobtained from Prof Ohsang Kown in the Department ofDermatology College of Medicine at Seoul National Uni-versity Human keratinocyte (HaCaT) cells were purchasedfrom American Type Culture Collection (ATCC) DPC isobtained from occipital scalp tissue containing more than100 hair follicles in twelve healthy male volunteers (20sim50ages Korean) according to the approval of Seoul NationalUniversity Hospital Institutional Review Board (IRB numberH-1112-096-390) Before starting this experiment the DPCwas cultured for three passages They were maintainedin DMEM supplemented with 10 FBS and 100120583gmLpenicillin-streptomycin-amphotericin B solution They weregrown asmonolayer cultures and kept at 37∘C in a humidifiedatmosphere with 5 CO

2for growth

24 MTT Assay Cell viability was measured by the MTTassay as previously described [24] Briefly the cells wereplated at a density of 4 times 104 cellswell in 24-well plates andcultured overnight in growth DMEM media After changinglow serum media (1 FBS) the cells were treated withvarious concentrations of RJ and incubated for 1 and 3 daysMTT solution (5mgmL) was then added to each well andincubated for additional 3 h at 37∘C Finally DMSO wasadded to solubilize the formazan salt and the amount offormazan generated was determined by measuring opticaldensity (OD) at 540 nm using GENios microplate spec-trophotometer (PowerWaveXS BioTek Instruments IncWinooski USA)

25 Western Blot Analysis Cells plated on microplate disheswere incubated with various concentrations of RJ and thenwashed with cold PBS The treated cells were collected byscraping with 300 120583L of RIPA buffer (iNtRON biotechnologySeongnam Korea) containing protease inhibitor cocktail(Thermo Fischer Scientific Pierce IL USA)The scraped cellswere allowed to lyse for additional 30min on ice with peri-odic vortexing Cell debris was removed by centrifugation(13000timesg at 4∘C for 30min) and the resulting supernatantwas collected and determined for its protein concentrationusing a Bio-Rad protein assay reagent (Bio-Rad CA USA)The samples were separated on 10 SDS-polyacrylamidegel and then transferred to PVDF membranes (Bio-RadCA USA) Western blot was probed with specific primaryantibodies overnight at 4∘C After washing the membraneswere incubated with horseradish peroxidase-conjugated sec-ondary antibody (Bethyl Laboratories Inc MontgomeryUSA) for 1 hr at room temperature The blots were visualizedby using an enhanced chemiluminescence method (ECLAmershamBiosciences Buckinghamshire UK) and analyzedusing ChemiDoc XRS (Bio-Rad CA USA) Densitometry

Evidence-Based Complementary and Alternative Medicine 3

analysis was performed with a Hewlett-Packard scanner andNIH Image software (Image J)

26 Immunofluorescence Analysis TheDPC and HaCaT cellswere grown on cell culture slide (SPL Life Science PocheonKorea) to 40ndash50 confluence After incubation for 24 h thecells were treated with RJ for another 24 h and then washedwith PBS three times for 5min The cells were fixed with 4paraformaldehyde for 10min and blocked with 3 FBS for1 h at room temperature (RT) Primary antibody (120573-catenin)was added to the cells and incubated overnight at 4∘C Thefollowingmorning the slides were washed three times in PBSand incubated with goat anti-rabbit immunoglobulin conju-gated with red-fluorescent dye for 1 h at RT Subsequentlythe stained cells were dropped with Vectashield mountingmedium containing DAPI to counterstain cell nuclei (VectorLaboratories CA USA) The samples were observed with aLeica fluorescence microscope (Leica Wetzlar Germany)

27 Experimental Study with RJ for Hair Growth This studywas performed by the Korea Institute of Toxicology (JinjuKorea) in accordance with the Institutional Animal Care andUse Committee (IACUC 1504-0003) 6-week-old C57BL6mice were purchased from Samtako Inc (Osan Korea) andadapted for 1weekThedorsal portion of 7-week-oldmicewasshaved with an animal clipper at which mouse hair follicleswere synchronized in the telogen stage Forty animals in 4randomized groups (119899 = 10) were used for studying thehair growing activity of RJ RJ was dissolved in a vehiclemixture (methanol PBS 60 40 vv)The animals in group1 were topically treated with vehicle mixture animals ingroups 2 and 3 were treated with RJ 4 and 8mgmL on dorsalskin once a day for 25 days Animals in group 4 (positivecontrol group) were treated with 5 minoxidil in the sameschedule with RJ treatment Animals were kept in isolationfor a certain amount of time and then housed back to separatecages To assess changes in hair morphology the mice wereanesthetized and photographed at 0 6 11 16 21 and 26days The ratio of hair growth (grown areashaved area) onthe photographs was analyzed using image analysis programNIS-Elements Basic Research Software (Nikon InstrumentsINC Tokyo Japan)

28 Histological Examination of Hair Follicles After 26 daysthemicewere sacrificed and their shaved skinswere collectedThe dorsal skin was cut into two parts for hematoxylinand eosin (HampE) staining and immunohistochemistry (IHC)uses The skin was fixed with 10 neutral formalin andembedded in paraffin blocks to obtain longitudinal andtransverse section 5120583m tissue sections were stained withhematoxylin and eosin (HampE) to confirm cycle of hair follicleThe percentage of hair follicles in each defined cycle stage atday 26 was calculated The other part of the tissue was frozenin liquid nitrogen for immunohistochemistry

29 Immunohistochemistry IHC for Ki-67 and120573-cateninwasperformed using rabbit monoclonal antibodies Immunore-activity was visualized with an avidin-biotin peroxidasereaction (PK-4001 Vectastain ABC kit) The peroxidase

reaction was developed using a 3 31015840-diaminobenzidinetetrahydrochloride (D-5905 Sigma)The sections were coun-terstained with hematoxylin before being mounted

210 Hair Morphological Change Using Scanning Electron Mi-croscope (SEM) The samples were placed on an adhesivecarbon tape attached to a metal stub and coated withplatinumusing a sputtering device Photomicrographs of hairfiber were obtained by a SEMusing JSM-7610F (JEOL TokyoJapan)

211 Statistical Analysis The results are expressed as meanplusmn standard deviation (SD) One-way analysis of variance(ANOVA) was used to evaluate the significance of differencebetween two mean values 119901 lt 001 was considered to bestatistically significant

3 Results

31 RJ Promotes DPC and HaCaT Cells Proliferation DPCand HaCaT cells were used as models in this study becausethey are important in the growth of hair To determinethe effects of RJ on DPC and HaCaT cells MTT assaywas performed In addition 100 120583M minoxidil (Mi) wasused as a positive control RJ enhanced the proliferation ofDPCs by 1321 and 1479 at the concentrations of 50 and100 120583gmL for 3 days compared with the control (Figure 1(a))In DPC treatment of RJ 100 120583gmL for 3 days exhibited thegreatest effectiveness which is stronger thanMi RJ treatmentsignificantly also promoted the proliferation of HaCaT cellsby 1313 at 50120583gmL for 3 days compared with the control(Figure 1(b)) The results suggest that RJ might have hairgrowth-promoting effect via the proliferation of DPC andHaCaT cells

32 RJ Activates Akt ERK12 andWnt120573-Catenin in DPC andHaCaT Cells Minoxidil has been well known to enhanceproliferation of hair cells through MAPK kinase family(ERK12 JNK12 and p38) and Akt signaling pathwaysTherefore the mechanism of proliferative effects by RJ inDPC and HaCaT cells was determined by western blot Asshown in Figure 2(a) the expression level of p-Akt wassteadily increased for up to 24 h in time-dependent manneron both DPC and HaCaT cells while p-ERK12 level waselevated up to 6 h in DPC and 1 h in HaCaT cells by treatmentof RJ 50120583gmL The phosphorylation of JNK12 and p38did not change in DPC and HaCaT cells (data not shown)Additionally RJ increased the expressions of 120573-catenin andp-GSK3120573 for 12 h in both DPC and HaCaT cells Treatmentwith dose-dependent manner of RJ increased the p-Aktand p-ERK12 as well as 120573-catenin and p-GSK3120573 in DPCand HaCaT cells for 24 h (Figure 2(b)) Nuclear 120573-cateninexpression also was increased by RJ treatment in both cells(Figure 2(c)) Interestingly the expression levels of 120573-cateninand p-GSK3120573 were greatly elevated by treatment with RJ butnot in Mi Moreover we confirmed significant activation of120573-catenin in DPC and HaCaT cells treated with RJ 50 120583gmLby immunofluorescence In control cells 120573-catenin was veryweakly detected in both cells However its expression was

4 Evidence-Based Complementary and Alternative Medicine

0 5 10 50 100 500 Mi

Cel

l via

bilit

y (

of c

ontro

l)

1 day3 days

0

20

40

60

80

100

120

140

160

180

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

lowast lowast

Concentrations (120583gmL)

(a)

0

20

40

60

80

100

120

140

160

Cel

l via

bilit

y (

of c

ontro

l)

0 5 10 50 100 500 Mi

1 day3 days

lowast lowastlowast lowast

lowast lowastlowast

lowast lowast

lowast

lowast

lowast

Concentrations (120583gmL)

(b)

Figure 1 Effect of RJ on proliferation in DPC and HaCaT cells (a) DPC and (b) HaCaT cells were treated with RJ (5 10 50 100 and500 120583gmL) for 1 and 3 days Cell viability was then determined in a MTT assay Minoxidil (Mi 100120583M) was used as positive control Thedata shown are the mean plusmn SD of three independent experiments Significant difference from control group lowast119901 lt 001

increased and detected in cytoplasm by treatment of RJ(Figures 2(d) and 2(e)) Similarly with western blotting resultexpression of 120573-catenin was increased by RJ in immunofluo-rescence data These results suggest that RJ can increase theproliferation of DPC and HaCaT cells via ERK12 Akt and120573-catenin signaling pathways

33 Regulation of RJ on Apoptosis-Related Proteins Since RJinduced the growth-promoting effect of DPC and HaCaTcells we examined the expression levels of the anti- andproapoptotic protein in the two Bcl-2 family proteins Inresponse to treatment of DPC with RJ the level of antiapop-totic Bcl-2 protein dose-dependently increased by 12- to 2-fold and the level of proapoptotic Bax protein significantlydecreased at only 50 120583gmL (Figure 3(a)) Additionally inHaCaT cells RJ induced significant upregulation of Bcl-2 pro-tein and downregulation of Bax protein in dose-dependentmanner (Figure 3(b)) Bcl-2Bax ratio was increased by 11- to24-fold and 12- to 22-fold in DPC and HaCaT cells as doseincreased from 125 to 50 120583gmL (Figures 3(a) and 3(b))

34 RJ Promotes Induction of Anagenic Phase in TelogenicC57BL6 Mice The black pigmentation was taken as evi-dence for transition of hair follicles from telogen to anagenphase To evaluate the hair growth-promoting activity of RJwe topically applied daily RJ onto the shaved back of telogenicC57BL6 mice for 25 days 5 Mi was separately applied asa positive control We evaluated the degree of hair growthby observing the skin color every 5 days At 11 days RJ-treated group showed partly black coloration and short hair

in the shaved skin while less visible black pigmentation wasobserved in vehicle group (Figure 4(a)) At 16 and 21 days RJ-treated group(8mgmL) showed markedly hair growth overhalf of mice At 26 days in RJ-treated group (8mgmL) themice backwas coveredwith overall hair except for threemicewhereas the vehicle group showed relatively less hair growthIn the Mi-treated group the change in the color of back skinwas earlier than in the other groups and covered completelyafter 26 days except for two mice The effect on hair growthof RJ treatment was assessed by measuring regrown area andshaved area Mi-treated group showed 839 of hair growthscore at 26 days compared with the vehicle group which keptshowing 26 of hair growth score Although less effectivethan Mi-treated group RJ-treated group exerted 399 and545 of hair growth score at 4 and 8mgmL at 26 days(Figure 4(b)) These results indicate that RJ promoted hairgrowth and telogen to anagen transition in C57BL6 mice

35 Effects of RJ on Development of Hair Follicle An increasein the number and the size of hair follicles during anageninduction is reported [25] To investigate whether RJ inducesthe anagen induction in hair follicle HampE staining wasperformed In the representative longitudinal sections thelength of hair follicle in RJ-treated group was longer andthe size was larger than in the vehicle group (Figure 5(a))Skin thickness also was increased in RJ- and Mi-treatedgroups compared to vehicle group Additionally as a result ofanalyzing hair cycle RJ-treated groups showed that anagenphase of hair follicle was increased over 2-fold and telogenphase was decreased by 6-fold as compared with vehicle

Evidence-Based Complementary and Alternative Medicine 5

DPCs0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(a)

DPCs

0 125 25 50 Mi 0 125 25 50 Mip-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(120583gmL) (120583gmL)

(b)

Lamin B

DPCs0 125 25 50

HaCaT cells(120583gmL)

Nuclear 120573-catenin

Lamin B

0 125 25 50 (120583gmL)

Nuclear 120573-catenin

(c)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(d)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(e)

Figure 2 Effect of RJ on ERK12 Akt p-GSK3120573 and120573-catenin expressions inDPC andHaCaT cells (a) Time-dependent effects of p-ERK12p-Akt p-GSK3120573 and 120573-catenin expressions were determined in cells treated with RJ (50120583gmL) for the indicated times (b) Dose-dependenteffects of p-ERK12 p-Akt p-GSK3120573 and 120573-catenin were detected in RJ-treated cells using western blotting (c) To confirm translocation of120573-catenin into nucleus nuclear 120573-catenin expression was determined by western blotting (d and e)The 120573-catenin expression was determinedby immunofluorescence staining with anti-120573-catenin in DPC and HaCaT cells

Bcl-2

Bax

0 125 25 50 Mi (120583gmL)

120573-Actin

DPCs cells

(a)

Bcl-2

Bax

HaCaT cells

0 125 25 50 Mi (120583gmL)

120573-Actin

(b)

Figure 3 Effects of RJ on expressions of Bcl-2 and Bax proteins (a) DPC and (b) HaCaT cells were treated with RJ (0 125 25 and 50120583gmL)The levels of Bcl-2 and Bax were analyzed by western blotting using specific monoclonal antibodies 120573-Actin was used as a loading controlThe data shown are the mean plusmn SD of six independent experiments Significant difference from control group lowast119901 lt 001

6 Evidence-Based Complementary and Alternative MedicineVe

hicle

5 M

i

Day 0 Day 11 Day 16 Day 21 Day 26Day 6

RJ4

mg

mL

RJ8

mg

mL

(a)

0

20

40

60

80

100

0 6 11 16 21 26

Scor

e of h

air g

row

th (

)

Day of treatment

VehicleMiRJ 4mgmLRJ 8mgmL

lowast

lowast

lowast lowast

lowast

lowast

(b)

Figure 4 Effect of RJ on the anagen induction in C57BL6 mice (a) After shaving the back skin was treated with vehicle RJ and 5 Mievery day for 25 daysThe back skin was photographed at 0 6 11 16 21 and 26 days after shaving (b) Morphological change in C57BL6 micetreated with vehicle RJ and Mi was analyzed Parts of the shaved and the hair growth area measured using the NIS-Elements Basic ResearchSoftware The data shown are the mean plusmn SD (119899 = 10) Significant difference from vehicle group lowast119901 lt 001

group The percentage ratio of anagen to telogen phase ofhair growth inRJ-treated group (8mgmL)was also increasedfrom 26 58 to 66 12 in comparison with vehicle group(Figure 5(b)) These results suggest that RJ enhanced the hairgrowth which appeared in the increase in the hair folliclenumber size length and skin thickness and induction ofearly telogen to anagen conversion compared to the vehiclegroup

36 Involvement between the Promoting Hair Growth Effectand 120573-Catenin To confirm the proliferation and promotingeffect in hair cells we performed the IHCanalysis usingKi-67which is cellular proliferationmarker [26] Immunoreactivity

to Ki-67 revealed that RJ 8mgmL and Mi enhanced morecellular proliferation within matrix part of hair follicles thanvehicle group (Figure 6) To understand the mechanism ofinducing anagen phase in RJ-treated group the expression of120573-catenin was confirmed using IHC analysis 120573-catenin pro-tein expression levels were strongly detected in RJ 8mgmLcompared to vehicle group (Figure 6)

4 Discussion

Hair loss is generally not a life-threatening disorder but it ispsychological disease that has an impact on social interactionTo date there are only two antihair loss drugs finasteride and

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

(a)

Telogen Anagen Catagen0

102030405060708090

100

Hai

r fol

licle

in h

air c

ycle

()

VehicleMi

lowast

lowast

lowast

lowast

lowast

RJ 4mgmLRJ 8mgmL

(b)

Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

analysis was performed with a Hewlett-Packard scanner andNIH Image software (Image J)

26 Immunofluorescence Analysis TheDPC and HaCaT cellswere grown on cell culture slide (SPL Life Science PocheonKorea) to 40ndash50 confluence After incubation for 24 h thecells were treated with RJ for another 24 h and then washedwith PBS three times for 5min The cells were fixed with 4paraformaldehyde for 10min and blocked with 3 FBS for1 h at room temperature (RT) Primary antibody (120573-catenin)was added to the cells and incubated overnight at 4∘C Thefollowingmorning the slides were washed three times in PBSand incubated with goat anti-rabbit immunoglobulin conju-gated with red-fluorescent dye for 1 h at RT Subsequentlythe stained cells were dropped with Vectashield mountingmedium containing DAPI to counterstain cell nuclei (VectorLaboratories CA USA) The samples were observed with aLeica fluorescence microscope (Leica Wetzlar Germany)

27 Experimental Study with RJ for Hair Growth This studywas performed by the Korea Institute of Toxicology (JinjuKorea) in accordance with the Institutional Animal Care andUse Committee (IACUC 1504-0003) 6-week-old C57BL6mice were purchased from Samtako Inc (Osan Korea) andadapted for 1weekThedorsal portion of 7-week-oldmicewasshaved with an animal clipper at which mouse hair follicleswere synchronized in the telogen stage Forty animals in 4randomized groups (119899 = 10) were used for studying thehair growing activity of RJ RJ was dissolved in a vehiclemixture (methanol PBS 60 40 vv)The animals in group1 were topically treated with vehicle mixture animals ingroups 2 and 3 were treated with RJ 4 and 8mgmL on dorsalskin once a day for 25 days Animals in group 4 (positivecontrol group) were treated with 5 minoxidil in the sameschedule with RJ treatment Animals were kept in isolationfor a certain amount of time and then housed back to separatecages To assess changes in hair morphology the mice wereanesthetized and photographed at 0 6 11 16 21 and 26days The ratio of hair growth (grown areashaved area) onthe photographs was analyzed using image analysis programNIS-Elements Basic Research Software (Nikon InstrumentsINC Tokyo Japan)

28 Histological Examination of Hair Follicles After 26 daysthemicewere sacrificed and their shaved skinswere collectedThe dorsal skin was cut into two parts for hematoxylinand eosin (HampE) staining and immunohistochemistry (IHC)uses The skin was fixed with 10 neutral formalin andembedded in paraffin blocks to obtain longitudinal andtransverse section 5120583m tissue sections were stained withhematoxylin and eosin (HampE) to confirm cycle of hair follicleThe percentage of hair follicles in each defined cycle stage atday 26 was calculated The other part of the tissue was frozenin liquid nitrogen for immunohistochemistry

29 Immunohistochemistry IHC for Ki-67 and120573-cateninwasperformed using rabbit monoclonal antibodies Immunore-activity was visualized with an avidin-biotin peroxidasereaction (PK-4001 Vectastain ABC kit) The peroxidase

reaction was developed using a 3 31015840-diaminobenzidinetetrahydrochloride (D-5905 Sigma)The sections were coun-terstained with hematoxylin before being mounted

210 Hair Morphological Change Using Scanning Electron Mi-croscope (SEM) The samples were placed on an adhesivecarbon tape attached to a metal stub and coated withplatinumusing a sputtering device Photomicrographs of hairfiber were obtained by a SEMusing JSM-7610F (JEOL TokyoJapan)

211 Statistical Analysis The results are expressed as meanplusmn standard deviation (SD) One-way analysis of variance(ANOVA) was used to evaluate the significance of differencebetween two mean values 119901 lt 001 was considered to bestatistically significant

3 Results

31 RJ Promotes DPC and HaCaT Cells Proliferation DPCand HaCaT cells were used as models in this study becausethey are important in the growth of hair To determinethe effects of RJ on DPC and HaCaT cells MTT assaywas performed In addition 100 120583M minoxidil (Mi) wasused as a positive control RJ enhanced the proliferation ofDPCs by 1321 and 1479 at the concentrations of 50 and100 120583gmL for 3 days compared with the control (Figure 1(a))In DPC treatment of RJ 100 120583gmL for 3 days exhibited thegreatest effectiveness which is stronger thanMi RJ treatmentsignificantly also promoted the proliferation of HaCaT cellsby 1313 at 50120583gmL for 3 days compared with the control(Figure 1(b)) The results suggest that RJ might have hairgrowth-promoting effect via the proliferation of DPC andHaCaT cells

32 RJ Activates Akt ERK12 andWnt120573-Catenin in DPC andHaCaT Cells Minoxidil has been well known to enhanceproliferation of hair cells through MAPK kinase family(ERK12 JNK12 and p38) and Akt signaling pathwaysTherefore the mechanism of proliferative effects by RJ inDPC and HaCaT cells was determined by western blot Asshown in Figure 2(a) the expression level of p-Akt wassteadily increased for up to 24 h in time-dependent manneron both DPC and HaCaT cells while p-ERK12 level waselevated up to 6 h in DPC and 1 h in HaCaT cells by treatmentof RJ 50120583gmL The phosphorylation of JNK12 and p38did not change in DPC and HaCaT cells (data not shown)Additionally RJ increased the expressions of 120573-catenin andp-GSK3120573 for 12 h in both DPC and HaCaT cells Treatmentwith dose-dependent manner of RJ increased the p-Aktand p-ERK12 as well as 120573-catenin and p-GSK3120573 in DPCand HaCaT cells for 24 h (Figure 2(b)) Nuclear 120573-cateninexpression also was increased by RJ treatment in both cells(Figure 2(c)) Interestingly the expression levels of 120573-cateninand p-GSK3120573 were greatly elevated by treatment with RJ butnot in Mi Moreover we confirmed significant activation of120573-catenin in DPC and HaCaT cells treated with RJ 50 120583gmLby immunofluorescence In control cells 120573-catenin was veryweakly detected in both cells However its expression was

4 Evidence-Based Complementary and Alternative Medicine

0 5 10 50 100 500 Mi

Cel

l via

bilit

y (

of c

ontro

l)

1 day3 days

0

20

40

60

80

100

120

140

160

180

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

lowast lowast

Concentrations (120583gmL)

(a)

0

20

40

60

80

100

120

140

160

Cel

l via

bilit

y (

of c

ontro

l)

0 5 10 50 100 500 Mi

1 day3 days

lowast lowastlowast lowast

lowast lowastlowast

lowast lowast

lowast

lowast

lowast

Concentrations (120583gmL)

(b)

Figure 1 Effect of RJ on proliferation in DPC and HaCaT cells (a) DPC and (b) HaCaT cells were treated with RJ (5 10 50 100 and500 120583gmL) for 1 and 3 days Cell viability was then determined in a MTT assay Minoxidil (Mi 100120583M) was used as positive control Thedata shown are the mean plusmn SD of three independent experiments Significant difference from control group lowast119901 lt 001

increased and detected in cytoplasm by treatment of RJ(Figures 2(d) and 2(e)) Similarly with western blotting resultexpression of 120573-catenin was increased by RJ in immunofluo-rescence data These results suggest that RJ can increase theproliferation of DPC and HaCaT cells via ERK12 Akt and120573-catenin signaling pathways

33 Regulation of RJ on Apoptosis-Related Proteins Since RJinduced the growth-promoting effect of DPC and HaCaTcells we examined the expression levels of the anti- andproapoptotic protein in the two Bcl-2 family proteins Inresponse to treatment of DPC with RJ the level of antiapop-totic Bcl-2 protein dose-dependently increased by 12- to 2-fold and the level of proapoptotic Bax protein significantlydecreased at only 50 120583gmL (Figure 3(a)) Additionally inHaCaT cells RJ induced significant upregulation of Bcl-2 pro-tein and downregulation of Bax protein in dose-dependentmanner (Figure 3(b)) Bcl-2Bax ratio was increased by 11- to24-fold and 12- to 22-fold in DPC and HaCaT cells as doseincreased from 125 to 50 120583gmL (Figures 3(a) and 3(b))

34 RJ Promotes Induction of Anagenic Phase in TelogenicC57BL6 Mice The black pigmentation was taken as evi-dence for transition of hair follicles from telogen to anagenphase To evaluate the hair growth-promoting activity of RJwe topically applied daily RJ onto the shaved back of telogenicC57BL6 mice for 25 days 5 Mi was separately applied asa positive control We evaluated the degree of hair growthby observing the skin color every 5 days At 11 days RJ-treated group showed partly black coloration and short hair

in the shaved skin while less visible black pigmentation wasobserved in vehicle group (Figure 4(a)) At 16 and 21 days RJ-treated group(8mgmL) showed markedly hair growth overhalf of mice At 26 days in RJ-treated group (8mgmL) themice backwas coveredwith overall hair except for threemicewhereas the vehicle group showed relatively less hair growthIn the Mi-treated group the change in the color of back skinwas earlier than in the other groups and covered completelyafter 26 days except for two mice The effect on hair growthof RJ treatment was assessed by measuring regrown area andshaved area Mi-treated group showed 839 of hair growthscore at 26 days compared with the vehicle group which keptshowing 26 of hair growth score Although less effectivethan Mi-treated group RJ-treated group exerted 399 and545 of hair growth score at 4 and 8mgmL at 26 days(Figure 4(b)) These results indicate that RJ promoted hairgrowth and telogen to anagen transition in C57BL6 mice

35 Effects of RJ on Development of Hair Follicle An increasein the number and the size of hair follicles during anageninduction is reported [25] To investigate whether RJ inducesthe anagen induction in hair follicle HampE staining wasperformed In the representative longitudinal sections thelength of hair follicle in RJ-treated group was longer andthe size was larger than in the vehicle group (Figure 5(a))Skin thickness also was increased in RJ- and Mi-treatedgroups compared to vehicle group Additionally as a result ofanalyzing hair cycle RJ-treated groups showed that anagenphase of hair follicle was increased over 2-fold and telogenphase was decreased by 6-fold as compared with vehicle

Evidence-Based Complementary and Alternative Medicine 5

DPCs0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(a)

DPCs

0 125 25 50 Mi 0 125 25 50 Mip-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(120583gmL) (120583gmL)

(b)

Lamin B

DPCs0 125 25 50

HaCaT cells(120583gmL)

Nuclear 120573-catenin

Lamin B

0 125 25 50 (120583gmL)

Nuclear 120573-catenin

(c)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(d)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(e)

Figure 2 Effect of RJ on ERK12 Akt p-GSK3120573 and120573-catenin expressions inDPC andHaCaT cells (a) Time-dependent effects of p-ERK12p-Akt p-GSK3120573 and 120573-catenin expressions were determined in cells treated with RJ (50120583gmL) for the indicated times (b) Dose-dependenteffects of p-ERK12 p-Akt p-GSK3120573 and 120573-catenin were detected in RJ-treated cells using western blotting (c) To confirm translocation of120573-catenin into nucleus nuclear 120573-catenin expression was determined by western blotting (d and e)The 120573-catenin expression was determinedby immunofluorescence staining with anti-120573-catenin in DPC and HaCaT cells

Bcl-2

Bax

0 125 25 50 Mi (120583gmL)

120573-Actin

DPCs cells

(a)

Bcl-2

Bax

HaCaT cells

0 125 25 50 Mi (120583gmL)

120573-Actin

(b)

Figure 3 Effects of RJ on expressions of Bcl-2 and Bax proteins (a) DPC and (b) HaCaT cells were treated with RJ (0 125 25 and 50120583gmL)The levels of Bcl-2 and Bax were analyzed by western blotting using specific monoclonal antibodies 120573-Actin was used as a loading controlThe data shown are the mean plusmn SD of six independent experiments Significant difference from control group lowast119901 lt 001

6 Evidence-Based Complementary and Alternative MedicineVe

hicle

5 M

i

Day 0 Day 11 Day 16 Day 21 Day 26Day 6

RJ4

mg

mL

RJ8

mg

mL

(a)

0

20

40

60

80

100

0 6 11 16 21 26

Scor

e of h

air g

row

th (

)

Day of treatment

VehicleMiRJ 4mgmLRJ 8mgmL

lowast

lowast

lowast lowast

lowast

lowast

(b)

Figure 4 Effect of RJ on the anagen induction in C57BL6 mice (a) After shaving the back skin was treated with vehicle RJ and 5 Mievery day for 25 daysThe back skin was photographed at 0 6 11 16 21 and 26 days after shaving (b) Morphological change in C57BL6 micetreated with vehicle RJ and Mi was analyzed Parts of the shaved and the hair growth area measured using the NIS-Elements Basic ResearchSoftware The data shown are the mean plusmn SD (119899 = 10) Significant difference from vehicle group lowast119901 lt 001

group The percentage ratio of anagen to telogen phase ofhair growth inRJ-treated group (8mgmL)was also increasedfrom 26 58 to 66 12 in comparison with vehicle group(Figure 5(b)) These results suggest that RJ enhanced the hairgrowth which appeared in the increase in the hair folliclenumber size length and skin thickness and induction ofearly telogen to anagen conversion compared to the vehiclegroup

36 Involvement between the Promoting Hair Growth Effectand 120573-Catenin To confirm the proliferation and promotingeffect in hair cells we performed the IHCanalysis usingKi-67which is cellular proliferationmarker [26] Immunoreactivity

to Ki-67 revealed that RJ 8mgmL and Mi enhanced morecellular proliferation within matrix part of hair follicles thanvehicle group (Figure 6) To understand the mechanism ofinducing anagen phase in RJ-treated group the expression of120573-catenin was confirmed using IHC analysis 120573-catenin pro-tein expression levels were strongly detected in RJ 8mgmLcompared to vehicle group (Figure 6)

4 Discussion

Hair loss is generally not a life-threatening disorder but it ispsychological disease that has an impact on social interactionTo date there are only two antihair loss drugs finasteride and

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

(a)

Telogen Anagen Catagen0

102030405060708090

100

Hai

r fol

licle

in h

air c

ycle

()

VehicleMi

lowast

lowast

lowast

lowast

lowast

RJ 4mgmLRJ 8mgmL

(b)

Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

0 5 10 50 100 500 Mi

Cel

l via

bilit

y (

of c

ontro

l)

1 day3 days

0

20

40

60

80

100

120

140

160

180

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

lowast lowast

Concentrations (120583gmL)

(a)

0

20

40

60

80

100

120

140

160

Cel

l via

bilit

y (

of c

ontro

l)

0 5 10 50 100 500 Mi

1 day3 days

lowast lowastlowast lowast

lowast lowastlowast

lowast lowast

lowast

lowast

lowast

Concentrations (120583gmL)

(b)

Figure 1 Effect of RJ on proliferation in DPC and HaCaT cells (a) DPC and (b) HaCaT cells were treated with RJ (5 10 50 100 and500 120583gmL) for 1 and 3 days Cell viability was then determined in a MTT assay Minoxidil (Mi 100120583M) was used as positive control Thedata shown are the mean plusmn SD of three independent experiments Significant difference from control group lowast119901 lt 001

increased and detected in cytoplasm by treatment of RJ(Figures 2(d) and 2(e)) Similarly with western blotting resultexpression of 120573-catenin was increased by RJ in immunofluo-rescence data These results suggest that RJ can increase theproliferation of DPC and HaCaT cells via ERK12 Akt and120573-catenin signaling pathways

33 Regulation of RJ on Apoptosis-Related Proteins Since RJinduced the growth-promoting effect of DPC and HaCaTcells we examined the expression levels of the anti- andproapoptotic protein in the two Bcl-2 family proteins Inresponse to treatment of DPC with RJ the level of antiapop-totic Bcl-2 protein dose-dependently increased by 12- to 2-fold and the level of proapoptotic Bax protein significantlydecreased at only 50 120583gmL (Figure 3(a)) Additionally inHaCaT cells RJ induced significant upregulation of Bcl-2 pro-tein and downregulation of Bax protein in dose-dependentmanner (Figure 3(b)) Bcl-2Bax ratio was increased by 11- to24-fold and 12- to 22-fold in DPC and HaCaT cells as doseincreased from 125 to 50 120583gmL (Figures 3(a) and 3(b))

34 RJ Promotes Induction of Anagenic Phase in TelogenicC57BL6 Mice The black pigmentation was taken as evi-dence for transition of hair follicles from telogen to anagenphase To evaluate the hair growth-promoting activity of RJwe topically applied daily RJ onto the shaved back of telogenicC57BL6 mice for 25 days 5 Mi was separately applied asa positive control We evaluated the degree of hair growthby observing the skin color every 5 days At 11 days RJ-treated group showed partly black coloration and short hair

in the shaved skin while less visible black pigmentation wasobserved in vehicle group (Figure 4(a)) At 16 and 21 days RJ-treated group(8mgmL) showed markedly hair growth overhalf of mice At 26 days in RJ-treated group (8mgmL) themice backwas coveredwith overall hair except for threemicewhereas the vehicle group showed relatively less hair growthIn the Mi-treated group the change in the color of back skinwas earlier than in the other groups and covered completelyafter 26 days except for two mice The effect on hair growthof RJ treatment was assessed by measuring regrown area andshaved area Mi-treated group showed 839 of hair growthscore at 26 days compared with the vehicle group which keptshowing 26 of hair growth score Although less effectivethan Mi-treated group RJ-treated group exerted 399 and545 of hair growth score at 4 and 8mgmL at 26 days(Figure 4(b)) These results indicate that RJ promoted hairgrowth and telogen to anagen transition in C57BL6 mice

35 Effects of RJ on Development of Hair Follicle An increasein the number and the size of hair follicles during anageninduction is reported [25] To investigate whether RJ inducesthe anagen induction in hair follicle HampE staining wasperformed In the representative longitudinal sections thelength of hair follicle in RJ-treated group was longer andthe size was larger than in the vehicle group (Figure 5(a))Skin thickness also was increased in RJ- and Mi-treatedgroups compared to vehicle group Additionally as a result ofanalyzing hair cycle RJ-treated groups showed that anagenphase of hair follicle was increased over 2-fold and telogenphase was decreased by 6-fold as compared with vehicle

Evidence-Based Complementary and Alternative Medicine 5

DPCs0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(a)

DPCs

0 125 25 50 Mi 0 125 25 50 Mip-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(120583gmL) (120583gmL)

(b)

Lamin B

DPCs0 125 25 50

HaCaT cells(120583gmL)

Nuclear 120573-catenin

Lamin B

0 125 25 50 (120583gmL)

Nuclear 120573-catenin

(c)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(d)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(e)

Figure 2 Effect of RJ on ERK12 Akt p-GSK3120573 and120573-catenin expressions inDPC andHaCaT cells (a) Time-dependent effects of p-ERK12p-Akt p-GSK3120573 and 120573-catenin expressions were determined in cells treated with RJ (50120583gmL) for the indicated times (b) Dose-dependenteffects of p-ERK12 p-Akt p-GSK3120573 and 120573-catenin were detected in RJ-treated cells using western blotting (c) To confirm translocation of120573-catenin into nucleus nuclear 120573-catenin expression was determined by western blotting (d and e)The 120573-catenin expression was determinedby immunofluorescence staining with anti-120573-catenin in DPC and HaCaT cells

Bcl-2

Bax

0 125 25 50 Mi (120583gmL)

120573-Actin

DPCs cells

(a)

Bcl-2

Bax

HaCaT cells

0 125 25 50 Mi (120583gmL)

120573-Actin

(b)

Figure 3 Effects of RJ on expressions of Bcl-2 and Bax proteins (a) DPC and (b) HaCaT cells were treated with RJ (0 125 25 and 50120583gmL)The levels of Bcl-2 and Bax were analyzed by western blotting using specific monoclonal antibodies 120573-Actin was used as a loading controlThe data shown are the mean plusmn SD of six independent experiments Significant difference from control group lowast119901 lt 001

6 Evidence-Based Complementary and Alternative MedicineVe

hicle

5 M

i

Day 0 Day 11 Day 16 Day 21 Day 26Day 6

RJ4

mg

mL

RJ8

mg

mL

(a)

0

20

40

60

80

100

0 6 11 16 21 26

Scor

e of h

air g

row

th (

)

Day of treatment

VehicleMiRJ 4mgmLRJ 8mgmL

lowast

lowast

lowast lowast

lowast

lowast

(b)

Figure 4 Effect of RJ on the anagen induction in C57BL6 mice (a) After shaving the back skin was treated with vehicle RJ and 5 Mievery day for 25 daysThe back skin was photographed at 0 6 11 16 21 and 26 days after shaving (b) Morphological change in C57BL6 micetreated with vehicle RJ and Mi was analyzed Parts of the shaved and the hair growth area measured using the NIS-Elements Basic ResearchSoftware The data shown are the mean plusmn SD (119899 = 10) Significant difference from vehicle group lowast119901 lt 001

group The percentage ratio of anagen to telogen phase ofhair growth inRJ-treated group (8mgmL)was also increasedfrom 26 58 to 66 12 in comparison with vehicle group(Figure 5(b)) These results suggest that RJ enhanced the hairgrowth which appeared in the increase in the hair folliclenumber size length and skin thickness and induction ofearly telogen to anagen conversion compared to the vehiclegroup

36 Involvement between the Promoting Hair Growth Effectand 120573-Catenin To confirm the proliferation and promotingeffect in hair cells we performed the IHCanalysis usingKi-67which is cellular proliferationmarker [26] Immunoreactivity

to Ki-67 revealed that RJ 8mgmL and Mi enhanced morecellular proliferation within matrix part of hair follicles thanvehicle group (Figure 6) To understand the mechanism ofinducing anagen phase in RJ-treated group the expression of120573-catenin was confirmed using IHC analysis 120573-catenin pro-tein expression levels were strongly detected in RJ 8mgmLcompared to vehicle group (Figure 6)

4 Discussion

Hair loss is generally not a life-threatening disorder but it ispsychological disease that has an impact on social interactionTo date there are only two antihair loss drugs finasteride and

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

(a)

Telogen Anagen Catagen0

102030405060708090

100

Hai

r fol

licle

in h

air c

ycle

()

VehicleMi

lowast

lowast

lowast

lowast

lowast

RJ 4mgmLRJ 8mgmL

(b)

Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

DPCs0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells0 1 3 6 12 24 (h)

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(a)

DPCs

0 125 25 50 Mi 0 125 25 50 Mip-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

HaCaT cells

p-Akt

p-ERK

120573-Catenin

p-GSK3120573

120573-Actin

(120583gmL) (120583gmL)

(b)

Lamin B

DPCs0 125 25 50

HaCaT cells(120583gmL)

Nuclear 120573-catenin

Lamin B

0 125 25 50 (120583gmL)

Nuclear 120573-catenin

(c)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(d)

DAPI Merge

Con

trol

RJ50120583

gm

L

120573-Catenin

(e)

Figure 2 Effect of RJ on ERK12 Akt p-GSK3120573 and120573-catenin expressions inDPC andHaCaT cells (a) Time-dependent effects of p-ERK12p-Akt p-GSK3120573 and 120573-catenin expressions were determined in cells treated with RJ (50120583gmL) for the indicated times (b) Dose-dependenteffects of p-ERK12 p-Akt p-GSK3120573 and 120573-catenin were detected in RJ-treated cells using western blotting (c) To confirm translocation of120573-catenin into nucleus nuclear 120573-catenin expression was determined by western blotting (d and e)The 120573-catenin expression was determinedby immunofluorescence staining with anti-120573-catenin in DPC and HaCaT cells

Bcl-2

Bax

0 125 25 50 Mi (120583gmL)

120573-Actin

DPCs cells

(a)

Bcl-2

Bax

HaCaT cells

0 125 25 50 Mi (120583gmL)

120573-Actin

(b)

Figure 3 Effects of RJ on expressions of Bcl-2 and Bax proteins (a) DPC and (b) HaCaT cells were treated with RJ (0 125 25 and 50120583gmL)The levels of Bcl-2 and Bax were analyzed by western blotting using specific monoclonal antibodies 120573-Actin was used as a loading controlThe data shown are the mean plusmn SD of six independent experiments Significant difference from control group lowast119901 lt 001

6 Evidence-Based Complementary and Alternative MedicineVe

hicle

5 M

i

Day 0 Day 11 Day 16 Day 21 Day 26Day 6

RJ4

mg

mL

RJ8

mg

mL

(a)

0

20

40

60

80

100

0 6 11 16 21 26

Scor

e of h

air g

row

th (

)

Day of treatment

VehicleMiRJ 4mgmLRJ 8mgmL

lowast

lowast

lowast lowast

lowast

lowast

(b)

Figure 4 Effect of RJ on the anagen induction in C57BL6 mice (a) After shaving the back skin was treated with vehicle RJ and 5 Mievery day for 25 daysThe back skin was photographed at 0 6 11 16 21 and 26 days after shaving (b) Morphological change in C57BL6 micetreated with vehicle RJ and Mi was analyzed Parts of the shaved and the hair growth area measured using the NIS-Elements Basic ResearchSoftware The data shown are the mean plusmn SD (119899 = 10) Significant difference from vehicle group lowast119901 lt 001

group The percentage ratio of anagen to telogen phase ofhair growth inRJ-treated group (8mgmL)was also increasedfrom 26 58 to 66 12 in comparison with vehicle group(Figure 5(b)) These results suggest that RJ enhanced the hairgrowth which appeared in the increase in the hair folliclenumber size length and skin thickness and induction ofearly telogen to anagen conversion compared to the vehiclegroup

36 Involvement between the Promoting Hair Growth Effectand 120573-Catenin To confirm the proliferation and promotingeffect in hair cells we performed the IHCanalysis usingKi-67which is cellular proliferationmarker [26] Immunoreactivity

to Ki-67 revealed that RJ 8mgmL and Mi enhanced morecellular proliferation within matrix part of hair follicles thanvehicle group (Figure 6) To understand the mechanism ofinducing anagen phase in RJ-treated group the expression of120573-catenin was confirmed using IHC analysis 120573-catenin pro-tein expression levels were strongly detected in RJ 8mgmLcompared to vehicle group (Figure 6)

4 Discussion

Hair loss is generally not a life-threatening disorder but it ispsychological disease that has an impact on social interactionTo date there are only two antihair loss drugs finasteride and

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

(a)

Telogen Anagen Catagen0

102030405060708090

100

Hai

r fol

licle

in h

air c

ycle

()

VehicleMi

lowast

lowast

lowast

lowast

lowast

RJ 4mgmLRJ 8mgmL

(b)

Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative MedicineVe

hicle

5 M

i

Day 0 Day 11 Day 16 Day 21 Day 26Day 6

RJ4

mg

mL

RJ8

mg

mL

(a)

0

20

40

60

80

100

0 6 11 16 21 26

Scor

e of h

air g

row

th (

)

Day of treatment

VehicleMiRJ 4mgmLRJ 8mgmL

lowast

lowast

lowast lowast

lowast

lowast

(b)

Figure 4 Effect of RJ on the anagen induction in C57BL6 mice (a) After shaving the back skin was treated with vehicle RJ and 5 Mievery day for 25 daysThe back skin was photographed at 0 6 11 16 21 and 26 days after shaving (b) Morphological change in C57BL6 micetreated with vehicle RJ and Mi was analyzed Parts of the shaved and the hair growth area measured using the NIS-Elements Basic ResearchSoftware The data shown are the mean plusmn SD (119899 = 10) Significant difference from vehicle group lowast119901 lt 001

group The percentage ratio of anagen to telogen phase ofhair growth inRJ-treated group (8mgmL)was also increasedfrom 26 58 to 66 12 in comparison with vehicle group(Figure 5(b)) These results suggest that RJ enhanced the hairgrowth which appeared in the increase in the hair folliclenumber size length and skin thickness and induction ofearly telogen to anagen conversion compared to the vehiclegroup

36 Involvement between the Promoting Hair Growth Effectand 120573-Catenin To confirm the proliferation and promotingeffect in hair cells we performed the IHCanalysis usingKi-67which is cellular proliferationmarker [26] Immunoreactivity

to Ki-67 revealed that RJ 8mgmL and Mi enhanced morecellular proliferation within matrix part of hair follicles thanvehicle group (Figure 6) To understand the mechanism ofinducing anagen phase in RJ-treated group the expression of120573-catenin was confirmed using IHC analysis 120573-catenin pro-tein expression levels were strongly detected in RJ 8mgmLcompared to vehicle group (Figure 6)

4 Discussion

Hair loss is generally not a life-threatening disorder but it ispsychological disease that has an impact on social interactionTo date there are only two antihair loss drugs finasteride and

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

(a)

Telogen Anagen Catagen0

102030405060708090

100

Hai

r fol

licle

in h

air c

ycle

()

VehicleMi

lowast

lowast

lowast

lowast

lowast

RJ 4mgmLRJ 8mgmL

(b)

Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

(a)

Telogen Anagen Catagen0

102030405060708090

100

Hai

r fol

licle

in h

air c

ycle

()

VehicleMi

lowast

lowast

lowast

lowast

lowast

RJ 4mgmLRJ 8mgmL

(b)

Figure 5 Analysis of histological change in C57BL6 mice treated with vehicle RJ and minoxidil for 25 days (a) Longitudinal sections ofthe dorsal skins were stained using HampE at 26 days (b) The cycle progression of hair follicle was analyzed by counting

minoxidil Those drugs have been used in clinical affairs buttheir efficacy is transient and they cause undesirable adverseeffects Therefore herbal plants with hair promoting activityin alternative medicine are increasingly attracting attentiondue to minimal or no side effect R japonicusHoutt has beentraditionally used to treat skin disease and problems in EastAsia [16] Recently some companies produce shampoo andsoap containing an extract of R japonicus Houtt and claimthat they are potential antihair loss products and promotehair growth effects However there is no scientific evidence toexplain these beneficial effects of RJ on hair growth Hence inthis study we investigated the hair growth-promoting effectof RJ using DPC HaCaT cells and C57BL6 mice

The DPC are major hair components and secrete anumber of growth factors that canmodulate the proliferationof follicular epithelium and follicle development [27] Severalstudies demonstrated that the size of DPC is well correlatedwith the hair growth and the number of DPC increases inthe early anagen stage [28 29] Keratinocyte plays pivotalroles in hair follicle and hair shaft The cells at early anagenare activated and subsequently fasted proliferating cells to

make hair bulb structure with hair matrix surrounding DPCbefore activation of hair follicle stem cell [30] MTT resultsindicated that RJ significantly stimulated proliferation by 13-fold in both DPC andHaCaT cells compared with nontreatedcells The proliferative effects of RJ on DPC and HaCaTcells are considered a maker maintenance and elongationof anagen phase In agreement with in vitro data the effectof RJ on hair growth was proved in in vivo study Theblack mouse C57BL6 is used for screening hair growthagents as an animal model for hair growth due to the factof producing their pigmentation during only anagen [31]The shaved back skins of C57BL6 were treated with topicalapplication of RJ (4 and 8mgmL) for 25 days RJ inducedstimulation of hair growth in the telogenic C57BL6 miceMice at higher dose group (RJ 8mgmL) showed significantand more abundant hair growth than those in the vehiclegroup and lower dose group (RJ 4mgmL) RJ-treated groupsalso showed that anagen phase was increased over 2-fold andtelogen phase was decreased by 6-fold resulting in that theratio of anagen to telogen phase was increased from 05 1 to55 1 inRJ-treated group 8mgmL In addition SEManalysis

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative MedicineKi

-67

120573-C

aten

in

Vehicle 5 MiRJ 4mgmL RJ 8mgmL

Figure 6 Effect of RJ on expression of hair growth-related protein Ki-67 (arrow) and 120573-catenin expression in the nuclei and cytoplasm ofproliferating follicular cells respectively (brown color) There are hairs with melanin pigment (black color) at the center of follicles

revealed that hair surface of RJ-treated groups is entirelyclear smooth and intact and is tightly overlapping cuticlewithout brokenness compared with vehicle group (see Sup-plement Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520161873746) Any adverse signs(skin irradiation drying and edema) were not observed atthe site of application of RJ for total experimental procedureFurthermore the histological data indicated that RJ-treatedgroup (8mgmL) showed increase in the number and the sizeof hair follicles skin thickness and an earlier induction ofthe anagen phase compared with vehicle group IHC analysisshowed that Ki-67 expression was upregulated in RJ 8mgmLgroup compared to that in vehicle group Collectively RJpromoted hair growth and anagen induction via proliferationof hair follicle-related cells and the length and size of hairfollicle in in vivo study

To elucidate the molecular mechanism underlying theability of RJ to induce hair growth- promoting effect theprotein levels ofMAPK kinase family and Akt which are hairfollicle cell proliferation-related molecules were checked inthe DPC and HaCaT cells Several reports demonstrated thathair growth agents promoted the proliferation and survivalof DPC through the activation of ERK12 and Akt [32ndash34]The ERK signaling is known to be activated bymitogens in alltypes of mammalian cells and its key roles in cell growth havebeen established previously [35] Furthermore it was recentlydemonstrated that ERK activation plays an important role

in the proliferation of DPC [33] Akt also mediates criticalsignals for cell survival and regulates the survival of DPCas antiapoptotic molecule [32] Western blot data showedthat the effect of RJ on hair growth was associated withactivation of ERK12 and Akt signaling RJ significantlyincreased ERK12 expression levels in both DPC and HaCaTcellsTherefore the activation of Akt by RJ might prolong thesurvival of DPC and HaCaT cells In addition expressions ofBcl-2 and Bax indicated that RJ prevented cell death in DPCand HaCaT cells

Recently among the development-related molecules inhair follicles it is known that 120573-catenin seems to be criticalin hair loss Several studies have suggested a relationshipbetween 120573-catenin signaling and alopecia When androgentreated DPC in androgenic alopecia patients 120573-catenin wassignificantly decreased in the cytoplasm while GSK-3120573 wasincreased [36 37] BIO (GSK-3120573 inhibitor) enhances hair-inducing ability through stabilization of 120573-catenin and ele-vated the alkaline phosphatase and insulin-growth factor-1 gene expressions [38 39] In addition 120573-catenin is keyregulator of hair follicle growth and cycling phase [40]During morphogenesis of hair follicle elevated expressionof 120573-catenin-dependent genes is observed Deletion of 120573-catenin inhibits formation of hair follicle while constitutivelyactivated 120573-catenin produces an expansion of hair folliclefate [41ndash43] 120573-catenin also induces the transition of hairfollicles from the telogen phase to anagen phase Hair cycle

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

regeneration and anagen phase maintenance are 120573-catenindependent [10 11] Therefore it was speculated whether RJinduced the hair growth-promoting effect through 120573-cateninpathway Western blot data showed that the expression levelsof 120573-catenin were upregulated by RJ-treated in DPC andHaCaT cells compared to those in the control or Mi-treatedcells IHC analysis results showed that the expression of 120573-catenin was increased in RJ-treated cells Furthermore inin vivo study there was a significant increase of 120573-cateninexpression in RJ-treated group (8mgmL) compared withvehicle group On the other hand it is recently reported thatactivation of ERK12 and Akt is related to 120573-catenin signal-ing pathway [44 45] Their phosphorylation increased 120573-cateninTCF complex through inactivation of GSK3120573 indi-cating that phosphorylation of ERK12 andor Akt inducesactivation of 120573-catenin signaling pathway Therefore theincrease of activation of ERK12 and Akt by treatment of RJmight help proliferation of hair cells as well as activation of 120573-catenin resulting in induction and maintenance of anagen

In conclusion although RJ has less hair growth effectcompared with minoxidil it stimulated hair growth byinducing anagen phase of hair follicles and proliferation ofhair cells We further showed new insights into possiblemechanism of action by which RJ may contribute to thepharmacological intervention of alopecia therapy Howeverthere is no doubt that the limitations of this study stillneed further investigations and improvements Additionalclinical trials and studies will be necessary to investigate whatcomponents in RJ contribute to its efficacy

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

ChangkeunKang conceived and designed the studyHyunky-oung Lee and Na-Hyun Kim mainly performed the exper-iments and wrote the manuscript Hyeryeon Yang SeongKyeongBae YunwiHeo InduChoudhary YoungChul KwonJae Kuk Byun Hyeong Jun Yim Byung Seung Noh andJeong-Doo Heo performed the in vivo study and analyzedthe data Jeong-Doo Heo and Euikyung Kim interpretedand discussed the data All authors have read and approvedthe final manuscript Hyunkyoung Lee and Na-Hyun Kimcontributed equally to this work

Acknowledgments

This study was supported by a grant sponsored by BiobeautyKorea Co Ltd (GNU 2015-0151) The authors would like tothank Prof Ohsang Kown the Department of DermatologyCollege of Medicine Seoul National University for kindlyproviding the human dermal papilla cells (DPCs)

References

[1] R Paus ldquoPrinciples of hair cycle controlrdquo The Journal ofDermatology vol 25 no 12 pp 793ndash802 1998

[2] Z S Goluch-Koniuszy ldquoNutrition of women with hair lossproblem during the period of menopauserdquo Przegląd Menopau-zalny vol 15 no 1 pp 56ndash61 2016

[3] N Hunt and S McHale ldquoThe psychological impact of alopeciardquoBritish Medical Journal vol 331 no 7522 pp 951ndash953 2005

[4] N Liu L-H Wang L-L Guo et al ldquoChronic restraint stressinhibits hair growth via substance p mediated by reactiveoxygen species in micerdquo PLoS ONE vol 8 no 4 Article IDe61574 2013

[5] E Perera L Yip and R Sinclair ldquoAlopecia areatardquo CurrentProblems in Dermatology vol 47 pp 67ndash75 2015

[6] K D Kaufman ldquoAndrogens and alopeciardquo Molecular andCellular Endocrinology vol 198 no 1-2 pp 89ndash95 2002

[7] J S Crabtree E J Kilbourne B J Peano et al ldquoA mouse modelof androgenetic alopeciardquo Endocrinology vol 151 no 5 pp2373ndash2380 2010

[8] M Ridanpaa R Fodde and M Kielman ldquoDynamic expressionand nuclear accumulation of 120573-catenin during the developmentof hair follicle-derived structuresrdquoMechanisms of Developmentvol 109 no 2 pp 173ndash181 2001

[9] J Kishimoto R E Burgeson and B A Morgan ldquoWnt signalingmaintains the hair-inducing activity of the dermal papillardquoGenes and Development vol 14 no 10 pp 1181ndash1185 2000

[10] H Shimizu and B A Morgan ldquoWnt signaling through the120573-catenin pathway is sufficient to maintain but not restoreanagen-phase characteristics of dermal papilla cellsrdquo Journal ofInvestigative Dermatology vol 122 no 2 pp 239ndash245 2004

[11] D Enshell-Seijffers C Lindon M Kashiwagi and B AMorgan ldquo120573-catenin activity in the dermal papilla regulatesmorphogenesis and regeneration of hairrdquo Developmental Cellvol 18 no 4 pp 633ndash642 2010

[12] D Van Mater F T Kolligs A A Dlugosz and E R FearonldquoTransient activation of 120573-catenin signaling in cutaneous ker-atinocytes is sufficient to trigger the active growth phase of thehair cycle in micerdquo Genes and Development vol 17 no 10 pp1219ndash1224 2003

[13] S K Jang S T Kim D I Lee et al ldquoDecoction and fermen-tation of selected medicinal herbs promote hair regrowth byinducing hair follicle growth in conjunction with wnts signal-ingrdquo Evidence-Based Complementary and Alternative Medicinevol 2016 Article ID 4541580 10 pages 2016

[14] E Y Lee E J Choi J A Kim et al ldquoMalva verticillata seedextracts upregulate the Wnt pathway in human dermal papillacellsrdquo International Journal of Cosmetic Science vol 38 no 2pp 148ndash154 2016

[15] T Tong N Kim and T Park ldquoTopical application of oleuropeininduces anagen hair growth in telogen mouse skinrdquo PLoS ONEvol 10 no 6 Article ID e0129578 2015

[16] H-S Lee S-K Kim J-B Han et al ldquoInhibitory effectsof Rumex japonicus Houtt on the development of atopicdermatitis-like skin lesions in NCNga micerdquo British Journal ofDermatology vol 155 no 1 pp 33ndash38 2006

[17] Y-P Li A Takamiyagi S T Ramzi and S Nonaka ldquoInhibitoryeffect of Rumex japonicus Houtt on the porphyrin photooxida-tive reactionrdquo The Journal of Dermatology vol 27 no 12 pp761ndash768 2000

[18] O P Zee D K Kim H C Kwon and K R Lee ldquoA new epoxy-naphthoquinol from Rumex japonicusrdquo Archives of PharmacalResearch vol 21 no 4 pp 485ndash486 1998

[19] S Guo B Feng R Zhu J Ma and W Wang ldquoPreparativeisolation of three anthraquinones from Rumex japonicus by

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

high-speed counter-current chromatographyrdquo Molecules vol16 no 2 pp 1201ndash1210 2011

[20] X Zhou L Xuan and S Zhang ldquoStudy on the chemicalconstituents from Rumex japonicusHouttrdquo Zhong Yao Cai vol28 no 2 pp 104ndash105 2005

[21] A A Elzaawely T D Xuan and S Tawata ldquoAntioxidant andantibacterial activities of Rumex japonicus HOUTT Aerialpartsrdquo Biological and Pharmaceutical Bulletin vol 28 no 12 pp2225ndash2230 2005

[22] D S Jang J M Kim J-H Kim and J S Kim ldquo24-nor-Ursane type triterpenoids from the stems of Rumex japonicusrdquoChemical and Pharmaceutical Bulletin vol 53 no 12 pp 1594ndash1596 2005

[23] Q-C Xie and Y-P Yang ldquoAnti-proliferative of physcion 8-O-120573-glucopyranoside isolated from Rumex japonicus Houtt onA549 cell lines via inducing apoptosis and cell cycle arrestrdquoBMC Complementary and Alternative Medicine vol 14 article377 2014

[24] H Lee J-S Kim and E Kim ldquoFucoidan from seaweed Fucusvesiculosus inhibits migration and invasion of human lungcancer cell via PI3K-Akt-mTOR pathwaysrdquo PLoS ONE vol 7no 11 article e50624 2012

[25] H Ogawa and M Hattori ldquoRegulation mechanisms of hairgrowthrdquo Current Problems in Dermatology vol 11 pp 159ndash1701983

[26] T Scholzen and J Gerdes ldquoThe Ki-67 protein from the knownand the unknownrdquo Journal of Cellular Physiology vol 182 no 3pp 311ndash322 2000

[27] X Rushan H Fei M Zhirong and W Yu-Zhang ldquoIdentifi-cation of proteins involved in aggregation of human dermalpapilla cells by proteomicsrdquo Journal of Dermatological Sciencevol 48 no 3 pp 189ndash197 2007

[28] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[29] S-S Rho S-J Park S-L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[30] V Greco T Chen M Rendl et al ldquoA two-step mechanism forstem cell activation during hair regenerationrdquo Cell Stem Cellvol 4 no 2 pp 155ndash169 2009

[31] P M Plonka D Michalczyk M Popik B Handjiski ASlominski and R Paus ldquoSplenic eumelanin differs from haireumelanin in C57BL6 micerdquo Acta Biochimica Polonica vol 52no 2 pp 433ndash441 2005

[32] J H Han O S Kwon J H Chung K H Cho H C Eunand K H Kim ldquoEffect of minoxidil on proliferation andapoptosis in dermal papilla cells of human hair folliclerdquo Journalof Dermatological Science vol 34 no 2 pp 91ndash98 2004

[33] W Li X-Y Man C-M Li et al ldquoVEGF induces proliferationof human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERKrdquo Experimental Cell Research vol318 no 14 pp 1633ndash1640 2012

[34] S Munkhbayar S Jang A-R Cho et al ldquoRole of arachidonicacid in promoting hair growthrdquo Annals of Dermatology vol 28no 1 pp 55ndash64 2016

[35] W Zhang and H T Liu ldquoMAPK signal pathways in the regula-tion of cell proliferation in mammalian cellsrdquo Cell Research vol12 no 1 pp 9ndash18 2002

[36] T Kitagawa K-I Matsuda S Inui et al ldquoKeratinocyte growthinhibition through the modification ofWnt signaling by andro-gen in balding dermal papilla cellsrdquo The Journal of ClinicalEndocrinology and Metabolism vol 94 no 4 pp 1288ndash12942009

[37] G J Leiros A I Attorresi and M E Balana ldquoHair folliclestem cell differentiation is inhibited through cross-talk betweenWnt120573-catenin and androgen signalling in dermal papilla cellsfrom patients with androgenetic alopeciardquo British Journal ofDermatology vol 166 no 5 pp 1035ndash1042 2012

[38] K Yamauchi and A Kurosaka ldquoInhibition of glycogen synthasekinase-3 enhances the expression of alkaline phosphatase andinsulin-like growth factor-1 in human primary dermal papillacell culture and maintains mouse hair bulbs in organ culturerdquoArchives of Dermatological Research vol 301 no 5 pp 357ndash3652009

[39] T Soma S Fujiwara Y Shirakata K Hashimoto and J Kishi-moto ldquoHair-inducing ability of human dermal papilla cells cul-tured underWnt120573-catenin signalling activationrdquo ExperimentalDermatology vol 21 no 4 pp 307ndash309 2012

[40] K S Stenn and R Paus ldquoControls of hair follicle cyclingrdquoPhysiological Reviews vol 81 no 1 pp 449ndash494 2001

[41] R DasGupta and E Fuchs ldquoMultiple roles for activatedLEFTCF transcription complexes during hair follicle devel-opment and differentiationrdquo Development vol 126 no 20 pp4557ndash4568 1999

[42] J Huelsken R Vogel B Erdmann G Cotsarelis and WBirchmeier ldquo120573-Catenin controls hair follicle morphogenesisand stem cell differentiation in the skinrdquo Cell vol 105 no 4pp 533ndash545 2001

[43] Y Zhang T Andl S H Yang et al ldquoActivation of 120573-cateninsignaling programs embryonic epidermis to hair follicle faterdquoDevelopment vol 135 no 12 pp 2161ndash2172 2008

[44] Z Freyberg S J Ferrando and J A Javitch ldquoRoles of theAktGSK-3 and Wnt signaling pathways in schizophrenia andantipsychotic drug actionrdquo American Journal of Psychiatry vol167 no 4 pp 388ndash396 2010

[45] S-Y Shin O Rath A Zebisch S-M ChooWKolch andK-HCho ldquoFunctional roles of multiple feedback loops in extracel-lular signal-regulated kinase and Wnt signaling pathways thatregulate epithelial-mesenchymal transitionrdquo Cancer Researchvol 70 no 17 pp 6715ndash6724 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom