ethanol extract of alismatis rhizome inhibits adipocyte … ·...

Research ArticleEthanol Extract of Alismatis rhizome Inhibits AdipocyteDifferentiation of OP9 Cells

Yeon-Ju Park1 Mi-Seong Kim1 Ha-Rim Kim12 Jeong-Mi Kim1 Jin-Ki Hwang1

Sei-Hoon Yang3 Hye-Jung Kim4 Dong-Sung Lee5 Hyuncheol Oh567 Youn-Chul Kim567

Do-Gon Ryu8 Young-Rae Lee129 and Kang-Beom Kwon128

1 Center for Metabolic Function Regulation Wonkwang University School of Medicine 460 Iksan-daero Iksan CityJeonbuk 570minus749 Republic of Korea

2 BK21 Plus program amp Department of Smart Life-Care Convergence Wonkwang University Graduate School460 Iksan-daero Iksan City Jeonbuk 570minus749 Republic of Korea

3 Department of Internal Medicine Wonkwang University School of Medicine 460 Iksan-daeroIksan City Jeonbuk 570minus749 Republic of Korea

4Department of Family Medicine The Catholic University of Korea Incheon St Maryrsquos Hospital 56 Dongsu-ro Bupyeong-guIncheon 403minus720 Republic of Korea

5Hanbang Body-Fluid Research Center Wonkwang University 460 Iksan-daero Iksan City Jeonbuk 570minus749 Republic of Korea6 Standardized Material Bank for New Botanical Drugs College of Pharmacy Wonkwang University460 Iksan-daero Iksan City Jeonbuk 570minus749 Republic of Korea

7 Institute of Pharmaceutical Research and Development College of Pharmacy Wonkwang University460 Iksan-daero Iksan City Jeonbuk 570minus749 Republic of Korea

8Department of Korean Physiology Wonkwang University School of Korean Medicine 460 Iksan-daero Iksan CityJeonbuk 570minus749 Republic of Korea

9Department of Oral Biochemistry and Institute of Biomaterials-Implant Wonkwang University School of Dentistry460 Iksan-daero Iksan City Jeonbuk 570minus749 Republic of Korea

Correspondence should be addressed to Young-Rae Lee mindyrwkuackr and Kang-Beom Kwon dessonwkuackr

Received 6 February 2014 Revised 7 May 2014 Accepted 8 May 2014 Published 9 June 2014

Academic Editor Ravirajsinh Jadeja

Copyright copy 2014 Yeon-Ju Park et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The rhizome of Alisma orientale (Alismatis rhizome) has been used in Asia for promoting diuresis to eliminate dampness from thelower-jiao and to expel heat In this study an ethanol extract of the rhizome ofAlisma orientale (AOE)was prepared and its effects onadipocyte differentiation ofOP9 cells were investigated TreatmentwithAOE in a differentiationmedium for 5 days resulted in dose-dependent inhibition of lipid droplet formation in OP9 cells Furthermore AOE significantly inhibited adipocyte differentiationby downregulating the expression of the master transcription factor of adipogenesis peroxisome proliferation-activity receptor 120574(PPAR120574) and related genes including CCAATenhancer binding protein 120573 (CEBP120573) fatty acid-binding protein (aP2) and fattyacid synthase (FAS) AOE exerted its inhibitory effects primarily during the early adipogenesis stage (days 1-2) at which time italso exerted dose-dependent inhibition of the expression of CEBP120573 a protein related to the inhibition of mitotic clonal expansionAdditionally AOE decreased the expression of autophagy-related proteins including beclin 1 and the autophagy-related genes(Atg) 7 and Atg12 Our results indicate that AOErsquos inhibitory effects on adipocyte differentiation of OP9 cells are mediated byreduced CEBP120573 expression causing inhibition of mitotic clonal expansion and autophagy

1 Introduction

Alismatis rhizome (AR) is the rhizome of Alisma orientale(Sam) Juzepzuk and belongs to the Alismataceae family AR

has been commonly used for the treatment of dampness-retention syndromes such as edema dysuria and diarrheain Asia Furthermore a number of experimental studies havereported its therapeutic potential as an anti-inflammatory

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 415097 9 pageshttpdxdoiorg1011552014415097

2 Evidence-Based Complementary and Alternative Medicine

[1 2] antiallergic [3 4] antibacterial [5] and antioxidant [6]agent However the effects of AR on adipocyte differentiationhave not yet been investigated

Obesity is a serious health problem and is related to thedevelopment of diseases such as type 2 diabetes dyslipi-demias atherosclerosis and even some cancers [7ndash10] Atthe cellular level obesity is characterized by increases in thenumber and volume of adipocytes the primary storage sitefor energy in animals and humans Adipogenesis is the pro-cess bywhich undifferentiated preadipocytes are converted tofully differentiated adipocytes [11] At the onset of adipocytedifferentiation the expression of CCAATenhancer bindingprotein 120573 (CEBP120573) and CEBP120575 is induced and thesetranscription factors are thought to mediate the expressionof peroxisome proliferator-activated receptor 120574 (PPAR120574) andCEBP120572 [12ndash14] The activation of CEBP120572 and PPAR120574 leadsto terminal differentiation through their subsequent trans-activation of adipocyte-marker genes such as adiponectinlipoprotein lipase (LPL) fatty acid-binding protein 2 (aP2)and fatty acid synthase (FAS) all of which are involved in lipidmetabolism [15]

Autophagy is a major evolutionarily conserved cytoplas-mic degradation pathway that has also been implicated inadipose tissue development [16ndash18] The genes encoding thebasic components of the autophagymachinery are namedAtg(autophagy-related) genes and autophagy-deficient MEFs(atg5minusminus atg7minusminus) exhibit markedly reduced efficiency duringadipogenesis [16 18]

Thus in the present study we set out to examine the anti-adipogenic actions of an extract of Alisma orientale (AOE)rhizomes and to study the mechanisms involved

2 Materials and Methods

21 Reagents OP9 cells were purchased from the AmericanType Culture Collection (Manassas VA USA) Minimumessential medium alpha (MEM120572) fetal bovine serum (FBS)Alexa Fluor 568 goat anti-rabbit IgG and BODIPY 493503dye were purchased from Invitrogen (Carlsbad CA USA)Insulin 3-isobutyl-1-methylxanthine (IBMX) dexametha-sone (DEXA) and Oil Red O dye were purchased fromSigmaChemical Co (St LouisMOUSA) Antibodies againstPPAR120574 CEBP120572 CEBP120573 cyclin A cyclin D1 cyclin D2beclin 1 ATG7 ATG13 LC3 insulin R 120573 and 120573-actin werepurchased from Santa Cruz Biotechnology (Santa CruzCA USA) Antibodies against extracellular signal-regulatedkinases 1 and 2 (ERK12) phospho-ERK12 protein kinase B(Akt) and phospho-Akt were obtained from Cell SignalingTechnology (Beverly MA USA) All of the chemicals usedwere of analytical grade

22 Preparation of Extracts Rhizomes of Alisma orientale(Alismataceae) were purchased in February 2010 from theUniversityOriental Herbal Drugstore Iksan Korea andwereidentified by Professor Youn-Chul Kim College of Phar-macy Wonkwang University (Korea) A voucher specimen(Number WP10-02-4) was deposited at the Herbarium ofthe College of Pharmacy Wonkwang University Dried and

pulverized rhizomes ofAlisma orientale (50 g) were extractedtwice with hot 70 ethanol (1 L) for 2 h at room temperatureand filtered with filter paper The filtrate was evaporated invacuo to produce a 70 ethanol extract (1215 g 243 ww)The 70 ethanol extract was suspended in distilled water(100mL) followed by filtrationThe residue derived from thefiltration was dissolved in hot ethanol and filtered again Thefiltrate was then evaporated in vacuo to obtain a standard-ized fraction of Alisma orientale extract (AOE NNMBS073600mg 12 ww) A 50mg of AOE powder was dissolvedin 1mL of DMSO for treatment with OP9 cells NNMBS073was deposited at the Standardized Material Bank for NewBotanical Drugs Wonkwang University

23 Cell Culture and Induction of Adipocyte Differentia-tion OP9 cells were cultured in MEM120572 containing 20FBS 2mM l-glutamine 100UmL penicillin and 100 120583gmLstreptomycin at 37∘C in a 5 CO

2

incubator To inducedifferentiation 1-day postconfluent preadipocytes were incu-bated in a differentiation medium containing 10 FBS05mM IBMX 025120583M DEXA 175 nM insulin 2mM l-glutamine 100UmL penicillin and 100120583gmL streptomycinfor 2 days The medium was then changed to MEM120572containing 10 FBS 2mM l-glutamine and 175 nM insulinand the cells were cultured for a further 3 days

24 Determination of Cell Viability Effects of AOE on OP9cell viability were determined using an established MTTassay Briefly cells were seeded in a 96-well dish and incu-bated at 37∘C for 24 h to allow attachment The attached cellswere kept untreated or treated with 10 20 and 40 120583gmLAOE for various time periods at 37∘C The cells were thenwashed with phosphate-buffered saline (PBS) prior to addingMTT (05mgmL PBS) and incubating at 37∘C for 30minFormazan crystals were dissolved with dimethyl sulfoxide(100 120583Lwell) and detected at OD

570

using an Emax EndpointELISA microplate reader (Molecular Devices SunnyvaleCA USA)

25 Oil Red O Staining After the induction of adipocytedifferentiation cellswerewashedwith cold PBS fixed at roomtemperature with 4 formalin for 1 h and then rinsed with60 isopropanol OP9 cells were stained at room temperaturewithOil RedO for 1 h andwashed 4 timeswith distilledwaterIntracellular Oil Red O dye was quantified by elution intoisopropanol and measuring the OD

500

26 Automated Image Acquisition and Processing After dif-ferentiation adipocytes were washed with cold PBS fixedat room temperature with 4 paraformaldehyde for 30minand then washed again 3 times with cold PBS Blockingbuffer was then added and incubated for 45min at roomtemperature to prevent nonspecific antibody binding PPAR120574or CEBP120573 antibodies were added and incubated overnightCells were then washed 3 times and incubated for 1 hwith BODIPY 493503 dye for lipid droplet staining DAPIfor nuclear staining and Alexa Fluor 568 goat anti-rabbitor anti-mouse IgG for PPAR120574 and CEBP120573 respectively

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers and probes for real-time quantitative PCR

Genes Primer sequence Accession no

PPAR120574 51015840-GAAAGACAACGGACAAATCACC-3101584051015840-GGGGGTGATATGTTTGAACTTG-31015840 NM 011146

CEBP120572 51015840-TTGTTTGGCTTTATCTCGGC-3101584051015840-CCAAGAAGTCGGTGGACAAG-31015840 NM 007678

FABP4 51015840-AGCCTTTCTCACCTGGAAGA-3101584051015840-TTGTGGCAAAGCCCACTC-31015840 NM 024406

FAS 51015840-TGATGTGGAACACAGCAAGG-3101584051015840-GGCTGTGGTGACTCTTAGTGATAA-31015840 NM 007988

HSL 51015840-GGAGCACTACAAACGCAACGA-3101584051015840-TCGGCCACCGGTAAAGAG-31015840 NM 010719

LPL 51015840-GGACGGTAACGGGAATGTATGA-3101584051015840-TGACATTGGAGTCAGGTTCTCTCT-31015840 NM 008509

GAPDH 51015840-CGTCCCGTAGACAAAATGGT-3101584051015840-TTGATGGCAACAATCTCCAC-31015840 NM 008084

Images were acquired on an ArrayScanTM VTi automatedmicroscopy and image analysis system (Cellomics Inc Pitts-burgh PA USA) Using this automated and highly sensitivefluorescence-imaging microscope with a 20x objective andsuitable filter sets the stained cells were identified with DAPIin fluorescence channel 1 BODIPY 493503 in channel 2and Alexa Fluor 568 in channel 3 respectively Arbitraryvalues for BODIPY CEBP120573 and PPAR120574 fluorescence werecalculated from the standard deviation of pixel intensityunder the DAPI channel reflecting the intact DNA contentof the cell

27 Quantitative Real-TimePolymerase ChainReaction (PCR)Total RNAwas extracted from cells using a FastPure RNA kit(TaKaRa Shiga Japan) The RNA concentration and puritywere determined by absorbance at 260280 nm cDNA wassynthesized from 1 120583g of total RNA using a PrimeScriptRT reagent kit (TaKaRa) Expression of mRNA related toadipocyte differentiation was determined by real-time PCRusing the ABI PRISM 7900 Sequence Detection Systemand SYBR Green I (Applied Biosystems Foster City CAUSA) The primer sequences used are listed in Table 1 Allresults were normalized to the GAPDH housekeeping geneto control for variation in mRNA concentrations Relativequantification was performed using the comparative ΔΔ119862

119905

method according to the manufacturerrsquos instructions

28 Western Blot Analysis OP9 cells were pretreated with40 120583gmL AOE for 1 h and then differentiated at 37∘CCells were lysed with ice-cold mammalian protein extrac-tion reagent (M-PER) (Pierce Biotechnology Rockford ILUSA) and the protein concentration in the lysate wasdetermined using the Bradford method Samples (20120583g)were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis with 10 acrylamide and transferred toHybond-P polyvinylidene fluoride membranes (GE Health-care Life Sciences Buckinghamshire UK) using a westernblot apparatus Each membrane was blocked for 2 h with 2bovine serum albumin or 5 skim milk and then incubated

overnight at 4∘C with 1 120583gmL of a 1 2000 dilution ofprimary antibody HRP-conjugated IgG (1 2000 dilution)was used as the secondary antibody Protein expression levelswere determined by signal analysis using an image analyzer(Fuji-Film Tokyo Japan)

29 Statistical Analysis Statistical analyses were performedusing analysis of variance with Duncanrsquos posttest 119875 values ofgt005 were considered to be statistically significant

3 Results

31 Effects of AOE on Adipocyte Differentiation To investi-gate the actions of AOE on adipocyte differentiation OP9preadipocytes were induced to differentiate between theabsence or presence of various concentrations of the extractWe observed that there were decreasing effects in lipidaccumulation as revealed by Oil Red O staining (Figures1(b) and 1(c)) Following on from these results we checkedwhether AOE might affect cell viability We found that indifferentiating cells treated for various periods with 20 or40 120583gmL AOE cytotoxicity was not apparent when com-pared to the control cells (Figure 1(a))

To investigate at which stage of the differentiation processAOE inhibits adipogenesis we treated the cells with AOE atdifferent times during the early (days 0ndash2) or late stages (days3ndash5) or for the entire (days 0ndash5) differentiation period Theformation of lipid droplets and accumulation of triglyceridein adipocytes were blocked in all treatment groups asrevealed by BODIPY staining (green) which is specific forintracellular lipids (Figures 2(a) and 2(b)) We also stainedPPAR120574 protein to determine the expression level (red) andfound it to be downregulated in all AOE-treated groups(Figures 2(a) and 2(c))

32 Effects of AOE onAdipocyte Differentiation-RelatedGenesAdipocyte differentiation is accompanied by increased


024 48 72 96

Incubation time (hour)0

20

40

60

80

100Vi

abili

ty (

)

20120583gmL40120583gmL

(a)

ND D5 10

20 40 N

(b)

ND D5 10 20 40 N0

1

2

3

4

5

Oil-

red

O (f

old)

lowast

AOE (120583gmL)

lowastlowast

(c)

Figure 1 Effects of AOE on viability and lipid accumulation in OP9 cells (a) Confluent OP9 cells were differentiated into adipocytes inmedium containing adipogenic inducers for varying time intervals in the presence or absence of AOE (20 and 40120583gmL) as describedin Materials and Methods Effects of AOE on cell viability were measured by MTT assay and data were represented as relative cell viabilityvalues ((b) and (c)) Cells were treated with adipogenic inducers to trigger differentiation into adipocytes in the presence or absence of variousconcentrations of AOE After 5 days of differentiation the cells were subjected to Oil Red O staining for a qualitative (b) and quantitative (c)comparison of intracellular lipid accumulation The bars represent fold increases compared with ND groups Data are presented as mean plusmnSD values of at least 3 independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 compared to the D5 group ND no differentiation D5 differentiationday 5 and N negative control (20 120583gmL Radix Astragali extract)

expression of various transcription factors and adipocyte-specific genes PPAR120574 and CEBP120572 are essential for ter-minal adipocyte differentiation and we found their expres-sion to be significantly decreased in all treatment groupsupon treatment with 40 120583gmL AOE (Figure 3) We fur-ther investigated whether AOE-induced reduction of PPAR120574and CEBP120572 levels regulated the expression of their targetgenes including adipocyte protein 2 (aP2) fatty acid syn-thase (FAS) hormone-sensitive lipase (HSL) and lipoproteinlipase (LPL) Treatment with AOE (40 120583gmL) significantlydecreased expression of each of these proteins at various timeintervals (Figure 3)

33 Effects of AOE on CEBP120573 Expression during the EarlyStage of Adipogenesis CEBP120573 is induced at a very early stageof adipogenesis and plays a crucial role in initiating the differ-entiation program by activating the expression of PPAR120574 and

CEBP120572 two key adipogenic transcription factors We foundthat CEBP120573 expression was significantly decreased duringthe early stage of adipogenesis (days 0ndash2) in OP9 adipocytestreated with either 20 or 40 120583gmL of AOE (Figures 4(a)and 4(b)) When growth-arrested preadipocytes were treatedwith adipogenic inducers during the early stage the num-ber of adipocytes increased by approximately twofold Thisresponse was significantly inhibited by treatment with AOEwith the number of AOE-treated OP9 adipocytes beingsimilar to that of the control group (Figure 4(c))

34 Effects of AOE on Cyclin and Autophagy-Related ProteinExpression To determine the signaling pathway throughwhich AOE inhibits clonal expansion during the early stageof adipogenesis cyclins A D1 and D2 ERK and Aktprotein expressions were examined As shown in Figures 5(a)and 5(b) cyclin D1 expression was decreased while cyclins


ND D 20 20 2040 40 40

0ndash2 3ndash5 0ndash5(120583gmL)

(day)

DAPI

BODIPY

PPAR120574

Merge

(a)

ND D 20 40 20 40 20 40 (120583gmL)0ndash2 0ndash5 (day)

2

4

6

8

0

10

12

14

3ndash5

Lipi

d ((in

tens

itytimes

105)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

1

2

3

4

0

5

6

7

PPA

R120574(in

tens

itytimes

104)

ND D 20 40 20 40 20 40 (120583gmL)

0ndash2 0ndash5 (day)3ndash5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

Figure 2 Effects of AOE on lipid droplet formation and PPAR120574 expression in OP9 cells OP9 cells were treated with adipogenic inducersto trigger differentiation into adipocytes AOE 20 (40 120583gmL) was then added during the early stage (0ndash2 days) late stage (3ndash5 days) or theentire period of differentiation (0ndash5 days) (a) After 5 days of differentiation immunohistochemical staining of OP9 cells was carried outusing a specific antibody to visualize PPAR120574 (red) BODIPY 493503 for lipid droplets (green) and DAPI to visualize nuclei (blue) (b) Totalcellular lipid droplet content was determined by averaging cytosolic BODIPY intensities of individual cells (c) PPAR120574 levels were determinedby averaging the intensities of nuclear antibody staining in individual cells Approximately 5000 cells were analyzed for lipid droplet contentand PPAR120574 levels Data are expressed mean plusmn SD values of at least 3 independent experiments lowastlowast

119875

lt 001 compared to the D group ND nodifferentiation D differentiation

A and D2 were not altered by treatment with 40 120583gmLAOE Furthermore ERK and AKT phosphorylations wereboth increased after treatment with adipogenic inducersfor 10min but treatment with AOE attenuated AKT butnot ERK phosphorylation Since autophagy is one of themajor factors involved in regulating the early events inadipocyte differentiation we also examined the expressionof autophagy-related proteins after 1 day of treatment withAOEWe found beclin1 Atg7 Atg12 and LC3 expression to beincreased after 1 day of treatment with adipogenic inducersHowever in cells treated with 40 120583gmL AOE activationof autophagy was significantly inhibited as indicated by

reduced levels of beclin1 Atg7 Atg12 and LC3 proteins(Figure 5(c))

4 Discussion and Conclusions

In the present study we show that AOE exerts anti-adipo-genic effects by a complex mechanism We found that AOEtreatment remarkably reduced levels of Oil Red O stainingin a concentration-dependent manner without affecting cellviability (Figure 1)

It is well known that when preadipocytes are induced todifferentiate the cells first initiate several rounds of mitotic


1

2

3

4

0

mRN

A (f

old)

PPRA120574

0ndash2 3ndash5 0ndash5ND D

Day

(a)

1

2

3

4

0

CEBP120572


Day

(b)

5

6

7

1

2

3

4

0

mRN

A (f

old)

aP2


Day

(c)

FAS

1

2

3

4


Day

(d)

mRN

A (f

old)

HSL


5

6

1

2

3

4

0

Day

(e)

LPL


5

6

7

1

2

3

4

0

Day

(f)

Figure 3 Effects of AOE on expression of PPAR120574 and PPAR120574-target genes OP9 cells were treated with adipogenic inducers to triggerdifferentiation into adipocytes AOE (40120583gmL) was added during the early stage (0ndash2 days) late stage (3ndash5 days) or for the entiredifferentiation period (0ndash5 days) After 5 days of differentiation real-time PCR was carried out using specific primers for PPAR120574 CEBP120572aP2 FAS HSL and LPL as described in Table 1 The genes transcript levels are expressed as ratios relative GAPDH expression with the levelin ND to 1 Data are expressed as mean plusmn SD values of at least 3 independent experiments ND no differentiation D differentiation

clonal expansion and then become quiescent while the coor-dinated transcription of adipogenic genes is initiated [19]Preadipocytes undergo mitotic clonal expansion throughupregulation of CEBP120573 and CEBP120575 during the early stageof adipocyte differentiation (day 0ndash2) [13] In this study

we found that during the early stage AOE inhibited lipiddroplet formation and suppressed CEBP120573 expression AOEalso inhibited adipocyte differentiation through suppressionof cell proliferation Cell proliferation during adipogenesisoccurs through the G

1

S checkpoint as shown by activation


ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)

Figure 4 Effects of AOE on CEBP120573 expression and cell proliferation in OP9 cells (a) OP9 cells were pretreated with 20 or 40120583gmL AOEfor 1 h and then cultured with adipogenic inducers After 1 day and 2 days of differentiation immunohistochemical staining of OP9 cellswas carried out using a specific antibody to visualize CEBP120573 (red) and DAPI to visualize nuclei (blue) (b) CEBP120573 expression levels weredetermined by averaging the nuclear antibody staining intensity from 5000 individual cells (c) The number of cells treated with 20 and40 120583gmL AOE was determined using a hemocytometer Experiments were carried out in triplicate and data are expressed as the mean plusmnSD values of at least 3 independent experiments lowast

119875

lt 005 lowastlowast 119875

lt 001 compared to the D group at days 1 and 2 respectively ND nodifferentiation D differentiation

of CDK2-cyclin EA and cyclin D1 turnover of P27kip1 andhyperphosphorylation of Rb protein [20] Furthermore thePI3KAkt pathway affects cell cycle progression throughregulation of cyclin D and P27kip1 expression [21] Here wefound that AOE treatment decreased cyclin D expression andphosphorylation of Akt in response to adipogenic inducersin OP9 cells (Figures 5(a) and 5(b)) In contrast whileadipogenic inducers also stimulate the MEKERK pathwayresulting in enhanced activity of CEBP120573 and inductionof adipocyte differentiation [22] AOE-treatment does notalter this response These results suggest that AOE mayexert its inhibitory effects on adipocyte differentiation viadownregulation of cyclin D1 and CEBP120573 expression whichin turn happens because of decreased Akt activation

PPAR120574 and CEBP120572 are both known to be directtranscriptional activators of adipocyte differentiation and

the most characterized adipocyte-specific regulatory DNAmotifs contain binding sites for both factors [23] PPAR120574is known to bind to the promoter region that induces theexpression of CEBP120572 which is in turn regulated byCEBP120573during adipocyte differentiation [24] CEBP120573 is expressedat earlier time points than both CEBP120572 and PPAR120574 duringadipogenesis [22] and it is known to induce the expressionof CEBP120572 and PPAR120574 [15] We found that AOE consid-erably reduces levels of CEBP120573 protein during the earlyphase (Figures 4(a) and 4(b)) and that it also significantlyinhibits expression of CEBP120572 and PPAR120574 at the mRNA level(Figure 3) It has been reported that adipogenesis-relatedgenes such as aP2 FAS HSL and LPL are targets of PPAR120574and CEBP120572 In our study AOE inhibited the expression ofeach of these adipocyte-specific genes (Figure 3) suggestingthat AOE inhibits the expression of CEBP120573 during the


Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)

Figure 5 Effects of AOE on cell proliferation and autophagy-related protein expression inOP9 cells OP9 cells pretreatedwith 40 120583gmLAOEwere then cultured with adipogenic inducers for the designated times After harvesting the lysates were subjected to western blot analysisfor cyclins A D1 and D2 (a) and ERK p-ERK Akt and p-Akt (b) and beclin1 Atg7 Atg12 and LC3B I and II (c) ND no differentiation Ddifferentiation D1 differentiation day 1

early stage of adipogenesis This in turn leads to reducedexpression of PPAR120574 and CEBP120572 ultimately leading toinhibition of lipid accumulation

A recent advance in understanding adipogenesis is therole played by autophagy a catabolic process for degradationof bulk cytoplasmic contents and subcellular organelles [25]Most of the genes involved in autophagy named autophagy-related genes (Atg) have been identified Importantly geneticdeletion of Atg5 and Atg7 two essential autophagy genessignificantly inhibits adipocyte differentiation in 3T3-L1 cellsand attenuates diet-induced obesity inmice [16 18]We foundthat after 1 day of differentiation levels of autophagy-relatedspecific proteins in OP9 cells such as beclin 1 Atg7 Atg12and LC3 were increased by adipogenic inducers Howeverin the AOE-treated groups increases in autophagy-relatedproteins were repressed (Figure 5(c))These findings demon-strate the functional importance of autophagy inhibitionin AOE-induced repression of adipocyte differentiation inOP9 cells Since early induction of both CEBP120573 expressionand autophagy is crucial for adipocyte differentiation it isplausible that the inhibition of autophagy induced by AOEmay be related to the decrease of CEBP120573 expression

In summary we have shown that AOE suppressesadipocyte differentiation in OP9 cells by downregulating theexpression of CEBP120573 and consequently decreasing PPAR120574and CEBP120573 levels AOE also inhibits adipogenesis by atten-uating autophagy in response to adipogenic inducers Thusour studies show that AOE exerts antiadipogenic actions and

therefore has promising therapeutic potential in preventingobesity

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yeon-Ju Park and Mi-Seong Kim contributed equally to thiswork

Acknowledgments

This research was supported by a National Research Founda-tion of Korea (NRF) Grant funded by the Korean Govern-ment (MEST) (no 2011-0030130) Republic of Korea and bya Basic Science Research Program Grant from the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education Science and Technology (NRF-2012R1A1A4A01011520)

References

[1] L Bravo ldquoPolyphenols chemistry dietary sources metabolismand nutritional significancerdquo Nutrition Reviews vol 56 no 11pp 317ndash333 1998


[2] Y Dai B Hang Z Huang and P Li ldquoAnti-inflammatory activ-ities and effect of rhizoma Alismatis on immune systemrdquoZhongguo Zhong Yao Za Zhi vol 16 no 10 pp 622ndash625 1991

[3] J H Lee O S Kwon H-G Jin E-R Woo Y S Kim and H PKim ldquoThe rhizomes of Alisma orientale and alisol derivativesinhibit allergic response and experimental atopic dermatitisrdquoBiological and Pharmaceutical Bulletin vol 35 no 9 pp 1581ndash1587 2012

[4] H Matsuda T Kageura I Toguchida T Murakami A Kishiand M Yoshikawa ldquoEffects of sesquiterpenes and triterpenesfrom the rhizomeofAlismaorientale onnitric oxide productionin lipopolysaccharide-activated macrophages absolute stere-ostructures of alismaketones-B 23-acetate and -C 23-acetaterdquoBioorganic and Medicinal Chemistry Letters vol 9 no 21 pp3081ndash3086 1999

[5] H-G Jin Q Jin A Ryun Kim et al ldquoA new triterpenoid fromAlisma orientale and their antibacterial effectrdquo Archives ofPharmacal Research vol 35 no 11 pp 1919ndash1926 2012

[6] C W Han E S Kang S A Ham H J Woo J H Lee andH G Seo ldquoAntioxidative effects of Alisma orientale extract inpalmitate-induced cellular injuryrdquo Pharmaceutical Biology vol50 no 10 pp 1281ndash1288 2012

[7] S D Hursting L M Lashinger L H Colbert et al ldquoEnergybalance and carcinogenesis underlying pathways and targetsfor interventionrdquo Current Cancer Drug Targets vol 7 no 5 pp484ndash491 2007

[8] D C W Lau B Dhillon H Yan P E Szmitko and S VermaldquoAdipokines molecular links between obesity and atheroslcero-sisrdquo American Journal of Physiology Heart and CirculatoryPhysiology vol 288 no 5 pp H2031ndashH2041 2005

[9] M W Rajala and P E Scherer ldquoMinireview the adipocytemdashat the crossroads of energy homeostasis inflammation andatherosclerosisrdquo Endocrinology vol 144 no 9 pp 3765ndash37732003

[10] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[11] T C Otto and M D Lane ldquoAdipose development from stemcell to adipocyterdquo Critical Reviews in Biochemistry and Molecu-lar Biology vol 40 no 4 pp 229ndash242 2005

[12] F M Gregoire C M Smas and H S Sul ldquoUnderstandingadipocyte differentiationrdquo Physiological Reviews vol 78 no 3pp 783ndash809 1998

[13] J M Ntambi and K Young-Cheul ldquoAdipocyte differentiationand gene expressionrdquo Journal of Nutrition vol 130 no 12 pp3122Sndash3126S 2000

[14] Q Tong and G S Hotamisligil ldquoMolecular mechanisms ofadipocyte differentiationrdquo Reviews in Endocrine and MetabolicDisorders vol 2 no 4 pp 349ndash355 2001

[15] S R Farmer ldquoTranscriptional control of adipocyte formationrdquoCell Metabolism vol 4 no 4 pp 263ndash273 2006

[16] R Baerga Y Zhang P-H Chen S Goldman and S JinldquoTargeted deletion of autophagy-related 5 (atg5) impairs adipo-genesis in a cellular model and in micerdquo Autophagy vol 5 no8 pp 1118ndash1130 2009

[17] M Tsukada and Y Ohsumi ldquoIsolation and characterizationof autophagy-defective mutants of Saccharomyces cerevisiaerdquoFEBS Letters vol 333 no 1-2 pp 169ndash174 1993

[18] Y Zhang S Goldman R Baerga Y Zhao M Komatsu andS Jin ldquoAdipose-specific deletion of autophagy-related gene 7(atg7) in mice reveals a role in adipogenesisrdquo Proceedings of the

National Academy of Sciences of the United States of Americavol 106 no 47 pp 19860ndash19865 2009

[19] O A MacDougald and M D Lane ldquoAdipocyte differentiationWhen precursors are also regulatorsrdquoCurrent Biology vol 5 no6 pp 618ndash621 1995

[20] K-M Choi Y-S Lee D-M Sin et al ldquoSulforaphane inhibitsmitotic clonal expansion during adipogenesis through cell cyclearrestrdquo Obesity vol 20 no 7 pp 1365ndash1371 2012

[21] R C Muise-Helmericks H L Grimes A Bellacosa S EMalstrom P N Tsichlis and N Rosen ldquoCyclin D expression iscontrolled post-transcriptionally via a phosphatidylinositol 3-kinaseAkt-dependent pathwayrdquo Journal of Biological Chem-istry vol 273 no 45 pp 29864ndash29872 1998

[22] Q-Q Tang T C Otto and M Daniel Lane ldquoMitotic clonalexpansion a synchronous process required for adipogenesisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 1 pp 44ndash49 2003

[23] U A White and J M Stephens ldquoTranscriptional factors thatpromote formation of white adipose tissuerdquo Molecular andCellular Endocrinology vol 318 no 1-2 pp 10ndash14 2010

[24] Y Hou P Xue Y Bai et al ldquoNuclear factor erythroid-derived factor 2-related factor 2 regulates transcription ofCCAATenhancer-binding protein120573 during adipogenesisrdquo FreeRadical Biology and Medicine vol 52 no 2 pp 462ndash472 2012

[25] B Levine and J Yuan ldquoAutophagy in cell death an innocentconvictrdquo Journal of Clinical Investigation vol 115 no 10 pp2679ndash2688 2005


[1 2] antiallergic [3 4] antibacterial [5] and antioxidant [6]agent However the effects of AR on adipocyte differentiationhave not yet been investigated

Obesity is a serious health problem and is related to thedevelopment of diseases such as type 2 diabetes dyslipi-demias atherosclerosis and even some cancers [7ndash10] Atthe cellular level obesity is characterized by increases in thenumber and volume of adipocytes the primary storage sitefor energy in animals and humans Adipogenesis is the pro-cess bywhich undifferentiated preadipocytes are converted tofully differentiated adipocytes [11] At the onset of adipocytedifferentiation the expression of CCAATenhancer bindingprotein 120573 (CEBP120573) and CEBP120575 is induced and thesetranscription factors are thought to mediate the expressionof peroxisome proliferator-activated receptor 120574 (PPAR120574) andCEBP120572 [12ndash14] The activation of CEBP120572 and PPAR120574 leadsto terminal differentiation through their subsequent trans-activation of adipocyte-marker genes such as adiponectinlipoprotein lipase (LPL) fatty acid-binding protein 2 (aP2)and fatty acid synthase (FAS) all of which are involved in lipidmetabolism [15]

Autophagy is a major evolutionarily conserved cytoplas-mic degradation pathway that has also been implicated inadipose tissue development [16ndash18] The genes encoding thebasic components of the autophagymachinery are namedAtg(autophagy-related) genes and autophagy-deficient MEFs(atg5minusminus atg7minusminus) exhibit markedly reduced efficiency duringadipogenesis [16 18]

Thus in the present study we set out to examine the anti-adipogenic actions of an extract of Alisma orientale (AOE)rhizomes and to study the mechanisms involved

2 Materials and Methods

21 Reagents OP9 cells were purchased from the AmericanType Culture Collection (Manassas VA USA) Minimumessential medium alpha (MEM120572) fetal bovine serum (FBS)Alexa Fluor 568 goat anti-rabbit IgG and BODIPY 493503dye were purchased from Invitrogen (Carlsbad CA USA)Insulin 3-isobutyl-1-methylxanthine (IBMX) dexametha-sone (DEXA) and Oil Red O dye were purchased fromSigmaChemical Co (St LouisMOUSA) Antibodies againstPPAR120574 CEBP120572 CEBP120573 cyclin A cyclin D1 cyclin D2beclin 1 ATG7 ATG13 LC3 insulin R 120573 and 120573-actin werepurchased from Santa Cruz Biotechnology (Santa CruzCA USA) Antibodies against extracellular signal-regulatedkinases 1 and 2 (ERK12) phospho-ERK12 protein kinase B(Akt) and phospho-Akt were obtained from Cell SignalingTechnology (Beverly MA USA) All of the chemicals usedwere of analytical grade

22 Preparation of Extracts Rhizomes of Alisma orientale(Alismataceae) were purchased in February 2010 from theUniversityOriental Herbal Drugstore Iksan Korea andwereidentified by Professor Youn-Chul Kim College of Phar-macy Wonkwang University (Korea) A voucher specimen(Number WP10-02-4) was deposited at the Herbarium ofthe College of Pharmacy Wonkwang University Dried and

pulverized rhizomes ofAlisma orientale (50 g) were extractedtwice with hot 70 ethanol (1 L) for 2 h at room temperatureand filtered with filter paper The filtrate was evaporated invacuo to produce a 70 ethanol extract (1215 g 243 ww)The 70 ethanol extract was suspended in distilled water(100mL) followed by filtrationThe residue derived from thefiltration was dissolved in hot ethanol and filtered again Thefiltrate was then evaporated in vacuo to obtain a standard-ized fraction of Alisma orientale extract (AOE NNMBS073600mg 12 ww) A 50mg of AOE powder was dissolvedin 1mL of DMSO for treatment with OP9 cells NNMBS073was deposited at the Standardized Material Bank for NewBotanical Drugs Wonkwang University

23 Cell Culture and Induction of Adipocyte Differentia-tion OP9 cells were cultured in MEM120572 containing 20FBS 2mM l-glutamine 100UmL penicillin and 100 120583gmLstreptomycin at 37∘C in a 5 CO

2

incubator To inducedifferentiation 1-day postconfluent preadipocytes were incu-bated in a differentiation medium containing 10 FBS05mM IBMX 025120583M DEXA 175 nM insulin 2mM l-glutamine 100UmL penicillin and 100120583gmL streptomycinfor 2 days The medium was then changed to MEM120572containing 10 FBS 2mM l-glutamine and 175 nM insulinand the cells were cultured for a further 3 days

24 Determination of Cell Viability Effects of AOE on OP9cell viability were determined using an established MTTassay Briefly cells were seeded in a 96-well dish and incu-bated at 37∘C for 24 h to allow attachment The attached cellswere kept untreated or treated with 10 20 and 40 120583gmLAOE for various time periods at 37∘C The cells were thenwashed with phosphate-buffered saline (PBS) prior to addingMTT (05mgmL PBS) and incubating at 37∘C for 30minFormazan crystals were dissolved with dimethyl sulfoxide(100 120583Lwell) and detected at OD

570

using an Emax EndpointELISA microplate reader (Molecular Devices SunnyvaleCA USA)

25 Oil Red O Staining After the induction of adipocytedifferentiation cellswerewashedwith cold PBS fixed at roomtemperature with 4 formalin for 1 h and then rinsed with60 isopropanol OP9 cells were stained at room temperaturewithOil RedO for 1 h andwashed 4 timeswith distilledwaterIntracellular Oil Red O dye was quantified by elution intoisopropanol and measuring the OD

500

26 Automated Image Acquisition and Processing After dif-ferentiation adipocytes were washed with cold PBS fixedat room temperature with 4 paraformaldehyde for 30minand then washed again 3 times with cold PBS Blockingbuffer was then added and incubated for 45min at roomtemperature to prevent nonspecific antibody binding PPAR120574or CEBP120573 antibodies were added and incubated overnightCells were then washed 3 times and incubated for 1 hwith BODIPY 493503 dye for lipid droplet staining DAPIfor nuclear staining and Alexa Fluor 568 goat anti-rabbitor anti-mouse IgG for PPAR120574 and CEBP120573 respectively













119905





3 Results





024 48 72 96


20

40

60

80

100Vi

abili

ty (

)

20120583gmL40120583gmL

(a)

ND D5 10

20 40 N

(b)

ND D5 10 20 40 N0

1

2

3

4

5

Oil-

red

O (f

old)

lowast

AOE (120583gmL)

lowastlowast

(c)







ND D 20 20 2040 40 40


(day)

DAPI

BODIPY

PPAR120574

Merge

(a)


2

4

6

8

0

10

12

14

3ndash5

Lipi

d ((in

tens

itytimes

105)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

1

2

3

4

0

5

6

7

PPA

R120574(in

tens

itytimes

104)

ND D 20 40 20 40 20 40 (120583gmL)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)


119875








1

2

3

4

0

mRN

A (f

old)

PPRA120574


Day

(a)

1

2

3

4

0

CEBP120572


Day

(b)

5

6

7

1

2

3

4

0

mRN

A (f

old)

aP2


Day

(c)

FAS

1

2

3

4


Day

(d)

mRN

A (f

old)

HSL


5

6

1

2

3

4

0

Day

(e)

LPL


5

6

7

1

2

3

4

0

Day

(f)




1



ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)


119875







Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References








































119905





3 Results





024 48 72 96


20

40

60

80

100Vi

abili

ty (

)

20120583gmL40120583gmL

(a)

ND D5 10

20 40 N

(b)

ND D5 10 20 40 N0

1

2

3

4

5

Oil-

red

O (f

old)

lowast

AOE (120583gmL)

lowastlowast

(c)







ND D 20 20 2040 40 40


(day)

DAPI

BODIPY

PPAR120574

Merge

(a)


2

4

6

8

0

10

12

14

3ndash5

Lipi

d ((in

tens

itytimes

105)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

1

2

3

4

0

5

6

7

PPA

R120574(in

tens

itytimes

104)

ND D 20 40 20 40 20 40 (120583gmL)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)


119875








1

2

3

4

0

mRN

A (f

old)

PPRA120574


Day

(a)

1

2

3

4

0

CEBP120572


Day

(b)

5

6

7

1

2

3

4

0

mRN

A (f

old)

aP2


Day

(c)

FAS

1

2

3

4


Day

(d)

mRN

A (f

old)

HSL


5

6

1

2

3

4

0

Day

(e)

LPL


5

6

7

1

2

3

4

0

Day

(f)




1



ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)


119875







Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References





























024 48 72 96


20

40

60

80

100Vi

abili

ty (

)

20120583gmL40120583gmL

(a)

ND D5 10

20 40 N

(b)

ND D5 10 20 40 N0

1

2

3

4

5

Oil-

red

O (f

old)

lowast

AOE (120583gmL)

lowastlowast

(c)







ND D 20 20 2040 40 40


(day)

DAPI

BODIPY

PPAR120574

Merge

(a)


2

4

6

8

0

10

12

14

3ndash5

Lipi

d ((in

tens

itytimes

105)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

1

2

3

4

0

5

6

7

PPA

R120574(in

tens

itytimes

104)

ND D 20 40 20 40 20 40 (120583gmL)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)


119875








1

2

3

4

0

mRN

A (f

old)

PPRA120574


Day

(a)

1

2

3

4

0

CEBP120572


Day

(b)

5

6

7

1

2

3

4

0

mRN

A (f

old)

aP2


Day

(c)

FAS

1

2

3

4


Day

(d)

mRN

A (f

old)

HSL


5

6

1

2

3

4

0

Day

(e)

LPL


5

6

7

1

2

3

4

0

Day

(f)




1



ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)


119875







Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References





























ND D 20 20 2040 40 40


(day)

DAPI

BODIPY

PPAR120574

Merge

(a)


2

4

6

8

0

10

12

14

3ndash5

Lipi

d ((in

tens

itytimes

105)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

1

2

3

4

0

5

6

7

PPA

R120574(in

tens

itytimes

104)

ND D 20 40 20 40 20 40 (120583gmL)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)


119875








1

2

3

4

0

mRN

A (f

old)

PPRA120574


Day

(a)

1

2

3

4

0

CEBP120572


Day

(b)

5

6

7

1

2

3

4

0

mRN

A (f

old)

aP2


Day

(c)

FAS

1

2

3

4


Day

(d)

mRN

A (f

old)

HSL


5

6

1

2

3

4

0

Day

(e)

LPL


5

6

7

1

2

3

4

0

Day

(f)




1



ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)


119875







Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References





























1

2

3

4

0

mRN

A (f

old)

PPRA120574


Day

(a)

1

2

3

4

0

CEBP120572


Day

(b)

5

6

7

1

2

3

4

0

mRN

A (f

old)

aP2


Day

(c)

FAS

1

2

3

4


Day

(d)

mRN

A (f

old)

HSL


5

6

1

2

3

4

0

Day

(e)

LPL


5

6

7

1

2

3

4

0

Day

(f)




1



ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)


119875







Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References





























ND D 20 40 20 40ND D

Day 1 Day 2

DAPI

CEBP120573

(120583gmL)

Merge

(a)

CEB

P120573

0

2

4

6

8

10

12

14

16

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

(inte

nsity

times10

4)

(b)

0

5

10

15

20

25

30

35

40

Cell

s (times10

4)

ND NDD D20 40 20 40 (120583gmL)

Day 1 Day 2

lowast

lowastlowast

lowast

lowastlowast

(c)


119875







Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References





























Cyclin A

Cyclin D1

Cyclin D2

120573-Actin

ND NDD D40 40 (120583gmL)

Day 1 Day 2

(a)

P-ERK

P-AKT

ERK

AKT

120573-Actin

ND NDD D40 40 (120583gmL)

10min 3h

(b)

Beclin 1

ND D1 40

ATG12

ATG7

LC3B I

(120583gmL)

120573-Actin

LC3B II

(c)










Acknowledgments


References




























ethanol extract of alismatis rhizome inhibits adipocyte … ·...

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