International Immunopharmacology 17 (2013) 1037–1043

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International Immunopharmacology

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Scopoletin suppresses IL-6 production from fibroblast-like synoviocytesof adjuvant arthritis rats induced by IL-1β stimulation

Yannong Dou a,1, Bei Tong a,1, Zhifeng Wei a, Ying Li a, Yufeng Xia b,⁎, Yue Dai a,⁎⁎a State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, Chinab Department of Chinese Materia Medica Analysis, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China

Abbreviations: IL, interleukin; AA, adjuvant arthritis;fibroblast-like synoviocytes;MAPK,mitogen-activated proC; ERK, extracellular signal-regulated kinase; CREB, cAprotein.⁎ Correspondence to: Y.F. Xia, Department of Chinese

Pharmaceutical University, 24 Tong Jia Xiang, Nanjing83271400; fax: +86 25 85301528.⁎⁎ Correspondence to: Y. Dai, Department of PharmacolChina Pharmaceutical University, 24 Tong Jia Xiang, Nanji83271400; fax: +86 25 85301528.

E-mail addresses: yfxiacpu@126.com (Y. Xia), yuedaic1 These authors equally contributed to this paper.

1567-5769/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.intimp.2013.10.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 July 2013Received in revised form 9 October 2013Accepted 14 October 2013Available online 26 October 2013

Keywords:ScopoletinInterleukin-6 productionFibroblast-like synoviocytesRheumatoid arthritis

Scopoletin, a coumarin compound naturally occurring in many medicinal plants, has previously beendemonstrated to ameliorate synovial inflammation and destruction of cartilage and bone in adjuvant arthritis(AA) rats. As interleukin (IL)-6 is critically involved in the initiation and development of rheumatoid arthritis(RA), the present study was performed to investigate the effect of scopoletin on IL-6 production fromfibroblast-like synoviocytes (FLS) to get insight into its anti-RA mechanisms. FLS were isolated from synovialmembrane tissues of AA rats, and stimulated with IL-1β (10 ng/mL). Scopoletin, at concentrations of 15, 30,and 60 μM, was shown to only moderately inhibit FLS proliferation, but dramatically reduce IL-6 production atboth mRNA and protein levels. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase,extracellular signal-regulated kinase (ERK), protein kinase C (PKC) and cAMP response element binding protein(CREB). These findings suggest that scopoletin exerts anti-RA action probably through suppressing IL-6production from FLS via MAPK/PKC/CREB pathways.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Rheumatoid arthritis (RA) is a systemic inflammatory disease ofunknown etiology [1]. Among a variety of cytokines implicated in thepathogenesis of RA, IL-6 is particularly important. Abnormal high levelof IL-6, detected in both serum and synovial fluid of RA patients [2],promotes differentiation and activation of fibroblast-like synoviocytes(FLS), and facilitates lymphocyte homing and activation, which resultin the release of a large amount of inflammatory cytokines and finallycause arthritis. Moreover, the process is recurrent and can finally leadto chronic inflammation. IL-6-deficient mice showed delayed onsetand reduced severity of collagen-induced arthritis [3]. It is wellrecognized that reducing the expression of IL-6 or inhibiting its activityis an effective intervention against acute and chronic inflammation in

RA, rheumatoid arthritis; FLS,tein kinase; PKC, protein kinaseMP response element binding

Materia Medica Analysis, China210009, China. Tel.: +86 25

ogy of Chinese Materia Medica,ng 210009, China. Tel.: +86 25

pu@hotmail.com (Y. Dai).

ghts reserved.

RA. Currently, the recombinant humanized anti-IL-6 receptor antibodyTocilizumab has obtained FDA approval and been listed to treat RA [4].

Scopoletin (6-methoxy-7-hydroxycoumarin) is a coumarin com-pound isolated from many medicinal plants, such as Erycibe obtusifolia,Aster tataricus and Foeniculum vulgare. These plants have been used intraditional Chinese medicines for the treatment of various rheumatoiddiseases with a long history [5]. A variety of bioactivities have beenobserved from this compound, including anti-inflammation, anti-allergy [6], and anti-angiogenesis activity [7]. Previously, we reportedthat scopoletin, injected intraperitoneally, could effectively decreasethe inflammatory degree of joints and significantly downregulate theoverexpression of inflammatory cytokines such as IL-6 in the synovialtissues of adjuvant arthritis (AA) rats [8]. However, the detailedmechanisms remain unclear.

In the present study, we investigated the suppressive effects ofscopoletin on the production of IL-6 from FLS, one of the main sourcecells of IL-6 in the synovium, and explored the underlyingmechanisms.

2. Materials and methods

2.1. Animals

Sprague-Dawley rats (male, 6–7 weeks old, 200 ± 20 g) werepurchased from the experimental animal center of China PharmaceuticalUniversity. All animals were kept under climate-controlled conditions.

1038 Y. Dou et al. / International Immunopharmacology 17 (2013) 1037–1043

All experiments were conducted under the guidelines of current ethicalregulations for institutional animal care and use at China PharmaceuticalUniversity.

2.2. Chemicals and reagents

Scopoletin was isolated from the stems of E. obtusifolia Benth. Thepurity was determined to be higher than 98% by normalization of thepeak area detected by high-performance liquid chromatography (HPLC)[8]. Dulbecco's Modified Eagle's Medium (DMEM) was purchased fromGibco Chemical Co. (St. Grand Island); 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from SigmaChemical Co. (St. Louis, MO, USA); Mycobacterium butyricum wasobtained from Becton Drive Co. Ltd. (New Jersey, USA); IL-1R and IL-6primers were synthesized by SBS Genetech Co. Ltd. (Shanghai, China);rat IL-1β was purchased from PeproTech Int. (Connecticut, USA); ratIL-6 enzyme-linked immunosorbent assay (ELISA) kit was purchasedfrom Biosouse Int. (Lot Number: S033104E, California, USA); anti-ERK1/2 rabbit polyclonal antibody, anti-p-ERK1/2 rabbit polyclonalantibody, anti-p38 rabbit polyclonal antibody, anti-p-p38 rabbitpolyclonal antibody, anti-p-PKC rabbit monoclonal antibody, anti-p-CREB rabbit monoclonal antibody, peroxidase-conjugated secondaryanti-rabbit antibodies, anti-glyceraldehyde-3-phosphate dehydrogenase(GAPDH) mouse monoclonal antibody and peroxidase-conjugatedsecondary anti-mouse antibody were purchased from Bioworld (LotNumber: 0802, Georgia, USA). The other chemicals and reagents usedin these experiments were of analytical grade.

2.3. Induction of AA

Arthritis was induced by an intradermal injection of 100 μL ofComplete Freund's adjuvant (FCA), containing 10 mg heat-inactiveBCG (80 °C, 1 h) in 1mL paraffin oil into the base of right hind paw ofrats on day (d) 0. Clinical signs such as swelling of feet, ear erythema,incapacity to bend the ankle and the presence of nodules at the baseof the tails and were observed by d 13.

2.4. Cell culture

The FLS were prepared from AA rats. In brief, at d 21 after arthritisinduction, rat synovial tissues were obtained, and washed twice withphosphate-buffered saline (PBS), and then minced the tissues into1.5 × 1.5mm pieces and digested in 1mL DMEM containing 10% (v/v)NCS at 37 °C, 5% CO2. After FLS were cultured in flasks until 90%confluence, non-adherent tissues were digested in serum-starvedDMEM containing 0.25% trypsin for 5–8 min and centrifuged at 100 gfor 5 min. Cells were washed extensively with 10% NCS-DMEM andcultured in 20 mL flat-bottomed culture bottles with 10% FCS-DMEM,100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C, 5% CO2. Thesynoviocytes between passages 3 and 6 were used in the experiments.

2.5. Cell viability assay

The cell viability was measured using the MTT assay. FLS(1 × 105 cells/mL) were plated into 96-well plates and incubated at37 °C and 5% CO2. Then, FLS were allocated to 5 groups: normal (FLSin medium), control (FLS in medium plus IL-1β (10ng/mL)), scopoletin(FLS in medium plus scopoletin (15, 30, 60μM) and IL-1β (10ng/mL)).After treatment for 20h, 20μL ofMTT (5mg/mL)was added to eachwellfollowed by incubation for 4 h. The supernatants were removed, and150 μL of DMSO was added to each well. The formazan dye crystalswere dissolved for 10min, and absorbances at 570nm were measuredwith a microplate reader.

2.6. Measurement of IL-6

FLS (1 × 105 cells/mL) were plated into 96-well plates for 2 h andincubated with various concentrations of IL-1β (0.01, 0.1, 1, 10,100ng/mL) at 37 °C and 5% CO2 for 24h. Then the culture supernatantswere collected, and the amounts of IL-6 were assayed by ELISAaccording to the manufacturer's instructions. The optical absorbancewas read with a microplate reader at 450 nm. The appropriateconcentration of IL-1β was selected for further studies.

FLS (1 × 105 cells/mL) were incubated with various concen-trations of scopoletin (15, 30, 60 μM) in the presence or absence ofIL-1β (10 ng/mL) at 37 °C and 5% CO2 for 24 h. Then the amounts ofIL-6 in the culture supernatants were assayed by ELISA.

2.7. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis

After treatment with scopoletin for different time, FLS were washedand harvested. Total RNAwas isolated using TRIzol reagent (Invitrogen,Garlsbad, CA, USA). Total RNA (2.5 μg) was converted to cDNA inaccordance with the instructions of M-MLV reverse transcriptase(Invitrogen, Garlsbad, CA, USA). 2 μL of the cDNA mixture was used forPCR [9]. The amplification was performed in a DNA thermal cyclerunder the following condition: 30 cycles at 94 °C for 30 s, 52 °C 1min,72 °C 1 min; 40 cycles at 94 °C for 30 s, 56 °C 1 min, 72 °C 1 min; and40 cycles at 94 °C for 30 s, 54 °C 1min, 72 °C 1min. Final extension wasperformed at 72 °C for 10min.

The PCR reactionwas conducted using the following primers:β-actin:sense primer 5′-ACA TCT GCT GGA AGG TGG AC-3′, antisense primer5′-GGT ACC ACC ATG TAC CCA GG-3′; IL-1R: sense primer 5′-TCT TCTGTT GGG TGT CTT T-3′, antisense primer 5′-GCT CTT ACT GGG TGTTCT AT-3′; and IL-6: sense primer 5′-TGC CTT CTT GGG ACT GAT-3′,antisense primer 5′-CTG GCT TTG TCT TTC TTG TT-3′; β-actin wasused as an internal control. The PCR products were electrophoresedon a 1.5% agarose gel and visualized by GoldView staining under UVtransmission [10].

2.8. Western blot analysis

After treatment with scopoletin for 12 h, FLS were washed threetimes with ice-cold PBS. The cells were lysed by lysis buffer (50 mMTris–HCl (pH 8.0), 150mM NaCl, 0.02% NaN3, 1% NP40) for 30min onice. The cytosolic fraction was obtained from the supernatants after9000 g centrifugation at 4 °C for 5min. The concentration of protein inthe supernatants was determined by Bradford assay [11].

Samples (40 μg of protein) were separated on a 10% SDS-PAGE andtransferred to nitrocellulose membranes. After being blocked with 10%nonfat milk in PBS-Tween 20 (0.1%, PBST) for 1h at room temperature,the membranes were washed three times with PBST buffer andincubated with monoclonal antibodies against rat anti-ERK1/2 rabbitpolyclonal antibody, anti-p-ERK1/2 rabbit polyclonal antibody, anti-p38 rabbit polyclonal antibody, anti-p-p38 rabbit polyclonal antibody,anti-p-PKC rabbit monoclonal antibody, anti-p-CREB rabbit monoclonalantibody, anti-GAPDH mouse monoclonal antibody in PBST containing1% nonfat milk for 1 h. Subsequently, they were washed three timeswith PBST buffer and incubated with peroxidase-conjugated secondaryantibodies for 1 h at room temperature and washed three times withPBST [12]. The membranes were incubated with ECL reagent for 4minand exposed to X-ray film for 5–10 min. The densitometry analysisof the blotting was performed using Image-Pro Plus software andGAPDH was used as the internal control.

2.9. Statistical analysis

Data were presented as means ± S.D. Statistical significance wasevaluated by one-way analysis of variance (ANOVA) followed by post

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hoc Tukey's test. It was considered to be statistically significant when pvalues b0.05.

3. Results

3.1. Effect of scopoletin on the viability of FLS

The cytotoxic effect of scopoletin was evaluated by conventionalMTT assay. FLS were cultured in 2% NCS-DMEM medium with variousconcentrations of scopoletin in the presence or absence of IL-1β(10 ng/mL) for 24 h. It was shown that scopoletin (15, 30, 60 μM) didnot display remarkable cytotoxicity on FLS (Fig. 1). These con-centrations were used as the test concentrations in the consequentexperiments.

Fig. 2. Effect of scopoletin on the production of IL-6 from FLS induced by IL-1β. (A) FLSwere treated with IL-1β (0, 0.01, 0.1, 1, 10, 100 ng/mL) for 24 h. IL-6 was measuredusing ELISA. Each value was the means ± S.D. of three independent experiments.*p b 0.05, **p b 0.01 vs. the group treated without IL-1β. (B) FLS were treated withscopoletin (15, 30, 60μM) in the presence or absence of IL-1β (10ng/mL) for 24h. Normal

3.2. Effect of scopoletin on the production of IL-6 from FLS induced by IL-1β

IL-6 is a significant factor in the generation and maintenance ofarthritic joint pain at acute and chronic stages of RA [13]. As shown inFig. 2A, IL-1β (0.01, 0.1, 1, 10, 100 ng/mL) induced IL-6 productionfrom FLS in a concentration dependent manner. High level of IL-6 wasproduced after FLS were stimulated with IL-1β (10 ng/mL). Theconcentration was therefore used for subsequent experiments. FLSwere cultured in 2% NCS-DMEM medium with various concentrationsof scopoletin for 24 h. The production of IL-6 was measured usingELISA. Scopoletin (15, 30, 60 μM) showed concentration-dependentinhibition of IL-6 production, and the inhibitory percentages were27.8%, 51.1% and 64.4%, respectively (Fig. 2B).

(FLS in medium, basal level of IL-6), control (FLS in medium plus IL-1β (10 ng/mL)). IL-6was measured using ELISA. Each value was the means ± S.D. of three independentexperiments. *p b 0.05, **p b 0.01 vs. control.

3.3. Effect of scopoletin on IL-1R and IL-6 mRNA expression in FLS induced

by IL-1β

The mRNA levels of IL-1R and IL-6 in FLS were detected by RT-PCR.After exposed to IL-1β (10 ng/mL) for 6 h, IL-6 mRNA levels in FLSreached a peak, but IL-1R mRNA expression did not alter (Fig. 3A).IL-1R and IL-6 mRNA levels were semiquantified, respectively(Fig. 3B and C). Data showed that IL-1β could induce the activation ofFLS and increase the secretion of IL-6 in FLS. However, these effectswere not through up-regulating autoreceptor (IL-1R) expression. FLSwere pretreated with various concentrations of scopoletin for 12 hand then exposed to IL-1β (10 ng/mL) for 6 h. Scopoletin (15, 30,60 μM) showed concentration-dependent inhibition of IL-6 mRNAexpression and no significant effect on IL-1R mRNA expression(Fig. 3D–F). Data suggested that the decreasing IL-6 secretion effect

Fig. 1. Effect of scopoletin on the viability of FLS. FLS were treated with scopoletin (15, 30,60 μM) in the presence or absence of IL-1β (10 ng/mL) for 24 h. Normal (FLS in medium),control (FLS inmediumplus IL-1β (10ng/mL)). The cell viability wasmeasured usingMTTassay. Each value was the means± S.D. of three independent experiments. **p b 0.01 vs.control.

of scopoletin in FLS stimulated by IL-1β was not through down-regulating IL-1R expression.

3.4. Effect of scopoletin on MAPK activation in FLS induced by IL-1β

In FLS fromAA rats, IL-1β (10ng/mL) stimulation resulted inmarkedphosphorylation of MAPKs. The time course of p-p38, p38, p-ERK, andERK in cytosols of FLS after exposed to IL-1β was showed in Fig. 4A.The phosphorylation of p38 and ERK reached peaks at 15–20min withno change of total p38 and ERK. FLS were preincubated with scopoletin(15, 30, 60 μM) for 12 h, and then exposed to IL-1β for 15 min. Theexpression of p-p38, p38, p-ERK, ERK from FLSwas analyzed bywesternblotting. Scopoletin (15, 30, 60 μM) showed concentration-dependentinhibition of the phosphorylation of p38 and ERK induced by IL-1β,and it did not affect the expression of total p38 and ERK (Fig. 4B). Thelevels of p-p38, p38, p-ERK, and ERK were semiquantified, respectively(Fig. 4C and D).

3.5. Effect of scopoletin on PKC activation in FLS induced by IL-1β

Another classical signal pathway of IL-1β is PKC pathway. IL-1β cansignificantly increase PKC activation [14]. In the present study, IL-1β(10 ng/mL) stimulation led to significant phosphorylation of PKC inFLS from AA rats with a peak time between 5 and 10min (Fig. 5A). FLSwere preincubated with scopoletin (15, 30, 60 μM) for 12 h, and thenexposed to IL-1β for 10 min. PKC in cytosols of FLS were analyzed bywestern blotting. Scopoletin (15, 30, 60 μM) showed concentration-dependent inhibition of PKC phosphorylation induced by IL-1β(Fig. 5C). p-PKC level was semiquantified (Fig. 5B and D).

Fig. 3. Effect of scopoletin on IL-1R and IL-6mRNA expression in FLS induced by IL-1β. (A) Time course of IL-1R and IL-6mRNA expression in cytosols of FLS after exposed to IL-1β (10ng/mL).(B, C) Ratios of IL-1R (or IL-6)/β-actin relative to the group treatedwith IL-1β for 0hwere quantified. Each valuewas themean±S.D. of three independent experiments. *pb0.05, **p b0.01vs. the group treatedwith IL-1β for 0h. (D) FLS were preincubatedwith scopoletin (15, 30, 60 μM) for 6h, IL-1R and IL-6mRNA in cytosols of FLS after exposed to IL-1β (10ng/mL)were analyzedwith RT-PCR. (E, F) IL-1R and IL-6mRNA levelswere semiquantified, respectively. Ratios of IL-1R (or IL-6)/β-actin relative to normal groupwere quantified. Each valuewasthe mean± S.D. of three independent experiments. *p b 0.05, **p b 0.01 vs. control.

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3.6. Effect of scopoletin on CREB activation in FLS induced by IL-1β

To confirm that CREB was activated by IL-1β in FLS, p-CREB levels inFLS cytosols were analyzed by western blotting. In FLS from AA rats,stimulation with IL-1β (10ng/mL) induced significant phosphorylationof CREB with a peak time at 20 min (Fig. 6A). FLS were preincubatedwith scopoletin for 12 h, and then exposed to IL-1β for 20 min.Scopoletin (15, 30, 60μM) showed concentration-dependent inhibitionof the phosphorylation of CREB induced by IL-1β (Fig. 6C). p-CREB levelwas semiquantified (Fig. 6B and D).

4. Discussion

Scopoletin is previously reported by our research group to havetherapeutic effect on adjuvant-induced arthritis in rats. It can alsoattenuate monosodium urate crystal-induced acute inflammation inmurine air pouch model, reduce the activation of macrophages andthe production of inflammatory mediators. In vitro, scopoletin can

inhibit lipopolysaccharide-induced production of proinflammatory cy-tokines, especially IL-6, from RAW 264.7 cells probably by suppressionof the activation of MAPK pathways [15]. It is suggested that scopoletinmight exert anti-RA action by repressing the production of pro-inflammatory cytokines. In the present study, we further investigatedthe effects of scopoletin on IL-6 secretion from FLS, key effector cells inarthritis pathogenesis [16]. It was shown that scopoletin substantiallyreduced IL-6 release from FLS stimulated with IL-1β. The findingsprovided, at least partially, a plausible explanation for the anti-RA actionof scopoletin.

IL-6, which may be produced by T lymphocytes, B lymphocytes,monocytes and fibroblasts in areas affected by inflammation, plays apivotal role in the local and systemic inflammation of RA [17]. It canfacilitate leukocyte recruitment, FLS differentiation and osteoclastactivation, which lead to chronic inflammation, pannus formation andcartilage degeneration [18]. The overproduction of IL-6 in RA may bedue to the functional IL-6 promoter-174 G/C polymorphism. Thepotential contributions made by the IL-6-174 G/C polymorphism to RA

Fig. 4. Effect of scopoletin on MAPK activation in FLS induced by IL-1β. (A) Time course of p-p38, p38, p-ERK, and ERK in cytosols of FLS after exposed to IL-1β (10 ng/mL). (B) FLS werepreincubatedwith scopoletin (15, 30, 60μM) for 12h, p-p38, p38, p-ERK, and ERK in cytosols of FLS after exposed to IL-1β (10ng/mL)were analyzed bywestern blotting. (C, D) p-p38, p38,p-ERK and ERK levels were semiquantified, respectively. GAPDH was used as the internal control. Then ratios of p-p38 (or p38, p-ERK, ERK)/GAPDH relative to normal group werequantified. Each value was the mean± S.D. of three independent experiments. *p b 0.05, **p b 0.01 vs. control.

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susceptibility have been examined in several studies [19]. Clinicalresearches showed that the promoter region of IL-6 is polymorphism(IL-6-174 G/C) in RA patients' joints, which leads to the enhancedsusceptibility to disease finally. IL-6 targeted therapy is meaningful tocontrol joint inflammation of RA.

Scopoletin could concentration-dependently suppress the productionof IL-6 in FLS. To clarify the molecular mechanisms of scopoletin, weinvestigated its influences on signaling pathways induced by IL-1β. Asreported, a variety of signal transductions participate in the productionof IL-6 in FLS after stimulationwith IL-1β. One pathway isMAPK, whosemembers (including ERK, SAPK/JNK and p38 MAPK) are highlyexpressed in synovial membranes of RA [20]. All of these members areimportant regulatory proteins and play a central role in the activationof FLS induced by IL-1β [16]. ERK, the first identified kinase in MAPKfamily, can be activated in response to growth factors, stressstimulation, bacterial products, inflammatory mediators and so on. Itis the key factor in cellular processes in inflammatory events. p38MAPK is especially relevant to human inflammatory disease and isactivated in the rheumatoid synovium. Many factors can activateintracellular p38 rapidly, such as proinflammatory cytokines, phys-iological stress and osmotic pressure, thus induce production ofcytokines (IL-6, TNF-α, IL-8, etc.). Inhibition of p38 phosphorylationcan obviously reduce gene expression of many inflammatory me-diators. In this study, the effects of scopoletin on MAPK signalingpathways in FLS were studied. Data showed that scopoletin markedlysuppressed the increased phosphorylation of ERK and p38 withoutimpact on total ERK and p38. It was suggested that scopoletin reducedIL-6 production in FLS probably through inhibiting the activation ofMAPK pathways.

PKC pathway may also participate in the regulation of IL-6 ex-pression induced by IL-1β [21]. It can mediate the activation of p38MAPK, and thus induce IL-6 production. The specific inhibitors of PKCcan reduce IL-6 secretion in human mast cells induced by IL-1 [22,23].In the current study, IL-1β stimulation resulted in marked phos-phorylation of PKC in FLS, and scopoletin showed significant inhibitionof PKC phosphorylation. It means that the suppression of PKC activationby scopoletin might contribute to its inhibition of IL-6 production in FLSinduced by IL-1β.

IL-6 gene 5′-end promoter region includes the consensus se-quences of NF-κB, NF-IL-6, CREB and AP-1. CREB, as a cAMP-responsive transcriptional factor, binds to the CRE region in the IL-6 gene promoter and regulates IL-6 production in cardiac fibroblasts[24]. The inhibitory effect of phenylarsine oxide on IL-6 productionmay be due to decreasing cAMP accumulation and CREB activation[25]. In addition, substantial evidence suggests that vasoactiveintestinal peptide can enhance CREB phosphorylation and increaseIL-6 expression in mouse calvarial osteoblasts. The mechanism is likelyto be dependent on cAMP/PKA/CREB activation [26]. Moreover, CREBcan activate p38 MAPK and cAMP/PKA pathways, sequentiallycontributing to the production of IL-6 [24]. These findings suggestthat the phosphorylation of CREB is substantially involved in theregulation of IL-6 production. In the present study, IL-1β stimulationinduced a marked phosphorylation of CREB in FLS, and scopoletinshowed concentration-dependent inhibition. It indicated that sco-poletin might interfere with IL-6 expression at transcription levelby affecting CREB.

In summary, the findings demonstrated that scopoletin couldsignificantly decrease the production of IL-6 in FLS from AA rats,

Fig. 5. Effect of scopoletin on PKC activation in FLS induced by IL-1β. (A) Time course of p-PKC in cytosols of FLS after exposed to IL-1β (10ng/mL) for indicated time. (B) p-PKC levelsweresemiquantified. GAPDHwas used as the internal control. Ratios of p-PKC/GAPDH relative to the group treatedwith IL-1β for 0minwere quantified. Each valuewas themean±S.D. of threeindependent experiments. *p b 0.05, **p b 0.01 vs. the group treated without IL-1β. (C) FLS were preincubated with scopoletin (15, 30, 60 μM) for 12 h, PKC in cytosols of FLSexposed to IL-1β (10ng/mL) was analyzed by western blotting. (D) p-PKC levels were semiquantified. GAPDHwas used as the internal control. Then the ratios of p-PKC/GAPDH relativeto normal group were quantified. Each value was the mean± S.D. of three independent experiments. *p b 0.05, **p b 0.01 vs. control.

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which provided a reasonable explanation for its inhibitory effects onchronic inflammation in RA. The underlying mechanisms responsiblefor the action of scopoletin probably involve the prevention of MAPK,PKC and CREB phosphorylation. The precise mechanisms of scopoletinneed further investigations.

Fig. 6. Effect of scopoletin on CREB activation in FLS induced by IL-1β. (A) Time course of p-CREwere semiquantified. GAPDHwas used as the internal control. Ratios of p-CREB/GAPDH relativeof three independent experiments. *p b 0.05, **p b 0.01 vs. the group treated without IL-1β. (C)exposed to IL-1β (10ng/mL)were analyzed bywestern blotting. (D) p-CREB levelswere semiqunormal group were quantified. Each value was the mean± S.D. of three independent experim

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (No. 30672471) and the Priority AcademicProgram Development of Jiangsu Higher Education Institutions,

B in cytosols of FLS after exposed to IL-1β (10ng/mL) for indicated time. (B) p-CREB levelsto the group treatedwith IL-1β for 0minwere quantified. Each value was themean±S.D.FLS were preincubated with scopoletin (15, 30, 60 μM) for 12h, p-CREB in cytosols of FLSantified. GAPDHwas used as the internal control. Then ratios of p-CREB/GAPDH relative toents. *p b 0.05, **p b 0.01 vs. control.

1043Y. Dou et al. / International Immunopharmacology 17 (2013) 1037–1043

and was partially funded by the Program for Changjiang Scholars andInnovative Research Team in University (PCSIRT-IRT1193).

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