European Journal of Pharmacology 636 (2010) 181–188

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European Journal of Pharmacology

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Immunopharmacology and Inflammation

Phytoestrogen bavachin mediates anti-inflammation targeting IκBkinase-IκBα-NF-κB signaling pathway in chondrocytes in vitro

Cheng-Chung Cheng a,1, Yi-Hsin Chen b,1, Wen-Liang Chang c, Shih-Ping Yang a, Deh-Ming Chang d,Jenn-Haung Lai d,⁎, Ling-Jun Ho b,⁎a Division of Cardiology, Department of Medicine, Tri-Service General Hospital, Taiwan, ROCb Division of Gerontology Research, Institute of Cellular and System Medicine, National Health Research Institute, Zhunan, Taiwan, ROCc School of Pharmacy, National Defense Medical Center, Taiwan, ROCd Division of Rheumatology/Immunology and Allergy, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC

⁎ Corresponding authors. Ho is to be contacted at DivInstitute of Cellular and System Medicine, National HeaTaiwan, ROC. Tel./fax: +886 2 87918382. Lai, Divisionand Allergy, Department of Medicine, Tri-Service GeneMedical Center, Taipei, Taiwan, ROC. Tel.: +886 2 8792

E-mail addresses: haungben@tpts5.seed.net.tw (J.-H(L.-J. Ho).

1 Both Cheng-Chung Cheng and Yi-Hsin Chen share e

0014-2999/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ejphar.2010.03.031

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 October 2009Received in revised form 26 February 2010Accepted 12 March 2010Available online 31 March 2010

Keywords:OsteoarthritisPhytoestrogenNF-κBChondrocyteBavachinChemokine

The pro-inflammatory cytokine interleukin-1beta (IL-1β) plays critical roles in pathogenesis of osteoar-thritis. Although estrogen is protective for cartilage in osteoarthritis patients, it also potentially increases therisk of stroke and cancer. Phytoestrogens acting as natural estrogen receptor modulators may serve asalternatives. This study aimed to identify medicinal phytoestrogens that preserve anti-inflammatoryproperty and may function as potential chondro-protective compounds. Both human chondrocytes andchondrocytic cell line CHON-002 were used for this study. Protein concentrations or expressions weremeasured by ELISA or Western blot, respectively. The DNA-binding activity and transcriptional activity oftranscription factors were evaluated by electrophoretic mobility shift assay and dual-luciferase reporterassay, respectively. Cell migration was analyzed by chemotaxis assays. We found that among screenedphytoestrogens, bavachin could potently decrease IL-1β-induced nuclear factor-kappa B (NF-κB) but notactivator protein-1 (AP-1) DNA-binding activity. Bavachin also inhibited IκBα degradation, increased nucleartranslocation of p65 and p50 as well as decreased IκBα kinase (IKK) activity. Furthermore, bavachininhibited IL-1β-induced chemokine production that resulted in reduced migration of THP-1 monocytic cells.Our results suggest that through decreasing IL-1β-induced activation of IKK-IκBα-NF-κB signaling pathway,bavachin potentially protects cartilage from inflammation-mediated damage in joints of osteoarthritispatients.

ision of Gerontology Research,lth Research Institute, Zhunanof Rheumatology/Immunologyral Hospital, National Defense7135; fax: +886 2 87927136.. Lai), lingjunho@nhri.org.tw

qual contributions.

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Osteoarthritis is a slowly destructive process of joints associatedwith the aging process. Although several factors can possibly causejoint destruction observed in osteoarthritis patients, the inflamma-tion-mediated damage of chondrocytes and their supporting matrixplays important roles causing osteoarthritis pathology (Herrero-Beaumont et al., 2009; Pelletier et al., 2001). In osteoarthritis joints,pro-inflammatory cytokines like interleukin-1beta (IL-1β) and tumornecrosis factor-alpha (TNF-α) secreted from the infiltrating inflam-matory cells and chondrocytes can be readily detected (Malemud,2004). In a commonly recognized osteoarthritis animal model by

transecting the anterior cruciate ligament in dogs, the expression ofIL-1β increases in both synovium and cartilage (Pelletier et al., 1998).Pro-inflammatory cytokines cause cartilage damage through modu-lating the expression of matrix metalloproteinases (MMPs), inducingproteoglycan degradation and causing cellular apoptosis (Chen et al.,2006; Kim et al., 2006). Aside from cytokines, chemokines induced inosteoarthritis joints are capable of recruiting leukocytes into inflamedareas that further exaggerates the inflammatory process (Mazzettiet al., 2004; Yuan et al., 2001). Similar to pathogenic effects ofcytokines, chemokines also induce production of MMPs in fibroblasts,cultured chondrocytes and cartilage explants (Borzi et al., 2000).

The nuclear factor-kappa B (NF-κB) proteins are a family ofubiquitously expressed transcription factors that play crucial roles inmost inflammatory responses (Roman-Blas and Jimenez, 2006). In restingstatus, NF-κB exists in the cytosol as an inactive form associated with theinhibitory protein IκBα (Hayden and Ghosh, 2008; Vallabhapurapu andKarin, 2009). When an extracellular stimulus induces phosphorylation ofthe inhibitory subunit and its subsequent degradation, the active complexofNF-κB translocates fromcytosol intonucleus and stimulates a variety ofinflammation-related genes. The phosphorylation of IκBα is mediated by

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the IκBαkinase (IKK) that consists of three subunits, including IKKα, IKKβand the regulatory subunit IKK-γ. Both IKKα and IKKβ preserving kinaseactivity are responsible for phosphorylating IκBα.

Osteoarthritis develops increasingly inwomenaftermenopause, andit is thought that estrogen reduction may be a risk factor. Althoughepidemiologic studies reveal inconsistent conclusions regarding thebenefit/risk of estrogen replacement therapy on osteoarthritis, moststudies support a conclusion that estrogen replacement therapy canreduce severity and prevalence of osteoarthritis in postmenopausalwomen (Alexandersen et al., 2009; Roman-Blas et al., 2009). In animalmodels, estrogen depletion increases the risk of articular cartilage toosteoarthritis changes and long-term estrogen replacement therapyhistologically reduces severity of cartilage lesions of naturally occurringosteoarthritis (Ham et al., 2002; Sniekers et al., 2008). Owing to trialsshowing increased incidence of stroke and cancer in women receivingprolonged estrogen replacement therapy, selective estrogen receptormodulators, including raloxifene, tamoxifen and phytoestrogens, havebeen suggested as potentially useful alternatives (Arjmandi et al., 2004;Christgau et al., 2004; Tsai and Liu, 1992).

Phytoestrogens are a group of plants-derived compounds that bindestrogen receptors and act as natural selective estrogen receptormodulators (Setchell, 2001). Because of co-existent agonistic andantagonistic properties toward estrogen receptor, phytoestrogens maybear less malignant tendency (Setchell, 2001). The Legminosae plant,Psoralea corylifolia, is widely distributed in Asian countries and its fruitsused as traditional Chinese medicines preserve anti-bacterial, anti-tumor, coronary artery-broadening and estrogen-like activities (Wanget al., 2001). In the present study, we show that a phytoestrogencompound, bavachin, extracted from P. corylifolia, effectively inhibitedIL-1β-induced NF-κB signaling events in chondrocytes.

2. Materials and methods

2.1. Reagents

The recombinant human IL-1βwas purchased from R & D (St. Paul,MN). ELISA kits for measuring chemokines, including regulated uponactivation, normal T cell expressed and secreted (RANTES), monocytechemoattractant protein 2 (MCP-2), macrophage inflammatoryprotein-1alpha (MIP-1α) and MIP-1β were purchased from R & D.The antibodies against p65, p50, IκBα, IKKα, IKKβ, phosphorylatedIKK (recognize both phosphorylated IKKα and IKKβ) or upstreamstimulatory factor-2 (USF-2) were purchased from Cell Signaling(Beverly, MA) or Santa Cruz Biotechnology. Unless specified, the restof the reagents were purchased from Sigma-Aldrich ChemicalCompany.

2.2. Preparation of chondrocytes

The cartilage from osteoarthritis patients who received total kneejoint replacement was obtained aseptically with prior approval of theInstitutional Review Board, Tri-Service General Hospital. The prepa-ration of chondrocytes from cartilage was performed according to ourprevious report (Ho et al., 2005). In brief, the full thickness articularcartilagewas removed from the underlying bone and cut into pieces ofaround 0.5 cm2. After enzymatic digestion with 2 mg/ml Pronase(Calbiochem, La Jolla, CA) in serum-free DMEM/antibiotics (Gibco-BRL, Gaithersberg, MD) for 1 h at 37 °C in 5% CO2 atmosphere, thespecimens were then digested with collagenase I at 0.25 mg/ml inDMEM medium containing 5% fetal bovine serum for overnight.Finally, the cells in monolayer culture were suspended in medium at6–8×106 cells/ml and added to T75flask plates. Cells weremaintainedin DMEM medium containing 10% fetal bovine serum and antibioticsfor 5–7 days before use. For extensive molecular analysis, thechondrocyte cell line CHON-002, derived from the long bone of an18-week-old female fetus, was purchased fromAmerican Type Culture

Collection (ATCC, clone number CRL-2847™). According to theprotocol, chondrocytes were infected by the defective retroviruscontaining human telomerase reverse transcriptase gene under G418selection. The primary cells after immortalization develop fibroblast-like morphology with expressions of collagen type I, collagen type X,and aggrecan but the expression of collagen type II is barely detectable(ATCC and our preliminary observations). Such changes in geneexpression patterns have been observed in other immortalizedchondrocyte cell lines (Robbins et al., 2000; Steimberg et al., 1999).

2.3. Extraction and preparation of phytoestrogen compounds

The detailed preparation of phytoestrogen compounds will beprovided upon request. In here, the preparation of bavachin wasdescribed. The dried and powdered seeds (9.0 kg) of P. corylifolia L.were extracted with acetone (30 L×4) at room temperature. Thecombined extracts were concentrated under reduced pressure to yield ablack syrup (2.2 kg). The acetone extract was dissolved in 95% aqueousmethanol (2 L), and then partitionedwith n-hexane (1:1, ×3) to give then-hexane-soluble fraction (970.8 g) and 95%methanol layer. The residualmethanol solution was concentrated in vacum to give a residue, whichwas partitioned between ethyl acetate and n-butanol (900 ml×3) to giveethyl acetate (708.9 g) andn-butanol-soluble fractions (14.4 g). The ethylacetate-soluble fraction was subjected to chromatography over silicondioxide (70–230 mesh, 5 kg) and eluted with CHCl3, CHCl3-methanol(95:5), CHCl3-methanol (90:10), CHCl3-methanol (80:20), andmethanol,gradient, to give five fractions. The third fraction was separated bySephadex LH-20 with methanol-CHCl3 (90:10) as eluent to give threesubfractions. The second sub-fraction was re-chromatographed overSephadex LH-20 with methanol-CHCl3 (90:10) as eluent, and thenseparated by preparative HPLC (C-18, 70% methanol/H2O) to givebavachin (670.0 mg). The structure of bavachin was identified bycomparison of its spectral data with literature values. With similarapproaches, other purified phytoestrogen compounds, including coryli-decain, corylin, isopsoralen, 8-prenyldaidzein, isobavachalcone, andbakuchiol isolated from P. corylifolia were readily prepared. Genisteinwas purchased from Sigma. The purified drugs were dissolved indimethylsulfoxide to make a 100 mM stock concentration. Definedconcentrations from 1 µM to 20 µM were used to test their effects indesigned experiments.

2.4. Preparation of cytoplasmic and nuclear extracts

Both cytoplasmic and nuclear extracts were prepared according toour published work (Ho et al., 2005). Briefly, treated cells were left at4 °C in 50 μl of buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mMMgCl2, 1 mM DTT, 1 mM PMSF, and 3.3 μg/ml aprotinin] for 15 minwith occasional gentle vortexing. The swollen cells were centrifugedat 20,000 g 3 min. After removal of the supernatants (cytoplasmicextracts), the pelleted nuclei was washed with 50 μl buffer A andsubsequently, the cell pellets were resuspended in 30 μl buffer C(20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,25% glycerol, 1 mM DTT, 0.5 mM PMSF, and 3.3 μg/ml aprotinin) andincubated at 4 °C for 30 min with occasional vigorous vortexing. Themixtures were then centrifuged at 20,000 g 20 min, and the super-natants were used as nuclear extracts.

2.5. Electrophoretic mobility shift assay (EMSA)

The EMSA was performed as detailed in our previous report (Hoet al., 2005). The oligonucleotides containing NF-κB binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′) and AP-1 binding site (5′-CGCTTGATGAGTCAGCCGGAA-3′) were purchased and used as DNAprobes (Promega, Mandison, WI). The DNA probes were radio-labeledwith [γ-32p]ATP using the T4 kinase according to manufacturer'sinstructions (Promega). For binding reaction, the radio-labeled probes

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were incubated with 5 μg of nuclear extracts. The binding buffercontained 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA,1 mM DTT, 1 mM MgCl2, 4% glycerol, and 2 μg poly(dI-dC). Thereaction mixtures were left at room temperature to precede bindingreaction 20 min. For supershift assays, different monoclonal anti-bodies were pre-incubatedwith nuclear extracts for 30 min before theaddition of radio-labeled probes. The final reaction mixtures wereanalyzed in a 6% non-denaturing polyacrylamide gel with 0.25× Tris-borate/EDTA as an electrophoresis buffer.

2.6. Western blot

ECL Western blot (Amersham-Pharmacia, Piscataway, NJ) wasperformed as described (Ho et al., 2005). Briefly, after extensive wash,cells were pelleted and resuspended in lysis buffer. After periodicvortexing, the mixture was centrifuged, the supernatant was collectedand protein concentrations were measured. Equal amounts of cellularextracts were analyzed in 10% SDS-polyacrylamide gel electrophoresisand transferred to the nitrocellulose filter. For immunoblotting, thenitrocellulose filter was incubated with TBS-T containing 5% nonfat milk(milk buffer) for 2 h, and then blotted with antisera against specificproteins for another 2 h at room temperature. After washing with milkbuffer twice, the filter was incubated with secondary IgG conjugated tohorseradish peroxidase at a concentration of 1:5000 for 30 min. The filterwas then incubated with the substrate and exposed to an X-ray film.

2.7. Dual-luciferase reporter assay

The transient transfection assays were performed according to ourprevious report (Huang et al., 2009). In brief, chondrocytes wereseeded one day before the transfection experiment at a density of5×105 cells/6-cm dish. FuGENE 6 transfection reagent (Roche) was usedto co-transfect 1 μg/ml of the reporter plasmid pNF-κB-luciferasewith theinternal control plasmid pTK-Renilla-luciferase (Promega) at a ratio of100:1 into the cells in serum- and antibiotic-free DMEM. Four h aftertransfection, the culture medium was replaced with fresh DMEMsupplemented with 2% fetal bovine serum. For luciferase assay, total celllysates were prepared and the firefly luciferase activity was analyzed andnormalized to Renilla luciferase activity according to the instructions(Promega).

2.8. Chemotaxis assay

Chemotaxis assays were similarly performed according to ourprevious report on dendritic cells. In brief, the lower chambers of atranswell cassette (Corning Costar) were loaded with supernatantscollected from IL-1β-treated chondrocytes in the presence or in theabsence of bavachin as indicated. The upper chambers were equallyloaded with 1×105 THP-1 cells. The incubation period was 2 h at 37 °C.After incubation, THP-1 cells migrating from the upper chambers to thelower chambers were counted by flow cytometry. The acquired eventsfor a fixed time period of 60 s in a FACScan were determined usingCellQuest software.

2.9. ELISA

Standard ELISA methods as described by the manufacturers wereused to measure RANTES, MCP-2, MIP1-α and MIP1-β proteinconcentrations. All determinants were performed in triplicate andexpressed as mean±S.D.

2.10. Statistical analysis

When necessary, the results were expressed as mean±S.D. PairedStudent's t-test was used to determine significance (Pb0.05 isconsidered to be significant).

3. Results

3.1. Effect of compounds purified from P. corylifolia on IL-1β-inducedNF-κB activation

Given the important role of NF-κB signaling pathway in IL-1β-mediated chondrocyte activation, we screened several phytoestrogencompounds purified from P. corylifolia to determine their NF-κB-suppressive activities. Because we also aimed at investigatingmolecular mechanisms, a chondrocyte cell line CHON-002 waspurchased from ATCC and used for most of this study. In Fig. 1A, weshow that IL-1β at a concentration of 5 ng/ml strongly induced NF-κBDNA-binding activity in CHON-002 cells (Fig. 1A). This bindingreaction was specific because wild-type but not mutant κB oligonu-cleotides competed with the NF-κB DNA-binding activity. Althoughthe fast-moving complex could also be successfully completed bywild-type κB oligonucleotides, its inconsistent induction by IL-1β andunaffected in supershift assays suggest itself as a non-specific complexrather than a NF-κB-specific DNA-binding complex. The componentsof NF-κB DNA-binding complex were analyzed with specific anti-bodies against two major NF-κB family proteins p65 and p50. Pre-incubation of nuclear extracts of IL-1β-stimulated cells with anti-p65or anti-p50 antibody caused slow migration of the complex (Fig. 1B).The results indicate that both p65 and p50 were in the DNA-bindingcomplex induced by IL-1β. To examine the anti-inflammatory effectsof phytoestrogens, we individually incubated chondrocytes withequal concentrations (10 μM) of several purified compounds from P.corylifolia or genistein (Fig. 1C) for 24 h before IL-1β stimulation. Theprepared nuclear extracts were analyzed for NF-κB DNA-bindingactivities. The results show that one of the screened compounds,bavachin (labeled as compound 6), potently decreased NF-κB DNA-binding activity induced by IL-1β (Fig. 1D).

3.2. Bavachin dose-dependently down-regulated NF-κB but not AP-1DNA-binding activity

Chondrocytes were pre-incubated for 24 h with various concen-trations of bavachin, and then were stimulated with or without IL-1β.The cells were collected and the nuclear extracts were prepared andanalyzed with EMSA. As shown in Fig. 2A, bavachin decreased IL-1β-induced NF-κB DNA-binding activity in a dose-dependent manner. Incontrast, bavachin did not affect IL-1β-induced AP-1 DNA-bindingactivity (Fig. 2B). The mild decrease of AP-1 DNA-binding activity inboth 1 and 5 μM concentrations of bavachin treatment was notconsistently seen in other two independent experiments usingdifferent donor cells (Fig. 2B and data not shown). At higherconcentrations, bavachin even enhanced AP-1 DNA-binding activitywith reasons not yet clear. To further investigate whether thedecrease of DNA-binding activity will also result in reduction oftranscriptional activation, we transiently transfected pNF-κB-lucifer-ase and pTK-Renilla-luciferase reporter plasmids into CHON-002 cells.Eight h after transfection, the cells were treated with bavachin atvarious dosages for another 24 h and then stimulated with IL-1β toinduce NF-κB transcriptional activity. Luciferase assays were thenperformed accordingly. The results indicate that bavachin at con-centrations from 5 to 20 μM significantly inhibited NF-κB transcrip-tional activity (Fig. 2C).

3.3. Bavachin inhibited IKK-IκBα-NF-κB signaling pathway

To determine whether the increased DNA-binding activity of NF-κB was due to increased accumulation of p65 and p50 subunits in thenucleus, we performed Western blot to measure the levels of thesetwo transcription factors in the cytosol and in the nucleus of the cells.CHON-002 cells were treated with 5 ng/ml IL-1β for various timepoints and the nuclear levels of p65 and p50 were examined. As

Fig. 1. Effects of phytoestrogens on IL-1β-induced NF-κB DNA-binding activity. CHON-002 cells at 1×106/ml were treated with 5 ng/ml IL-1β for 2, 4, 6 or 8 h. After treatment, thecells were collected and nuclear extracts were prepared and analyzed by EMSA. To determine the specificity of NF-κB DNA-binding complex, the wild-type (Wt) or mutant (Mt) κboligonucleotides were pre-incubated with the nuclear extract for 30 min before adding radio-labeled κb oligonucleotides (A). In (B), the monoclonal antibodies against p65, p50 orIκBαwere pre-incubated with the nuclear extract for 30 min before adding the radiolabled κb oligonucleotides for EMSA analysis. SS: supershifts; NS: non-specific. Structures of thepurified phytoestrogens examined and genistein were shown in (C). In (D), chondrocytes were pre-incubated with 10 μM concentration of several purified phytoestrogensindividually for 24 h and then stimulated with IL-1β for another 2 h and nuclear extracts were prepared for EMSA analysis. Compound 1: corylidecain, 2: corylin, 3: isopsoralen, 4: 8-prenyldaidzein, 5: isobavachalcone, 6: bavachin, 7: bakuchiol and 8: genistein. The representative results from at least three independent experiments are shown.

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shown in Fig. 3A, increased levels of p65 and p50 could be detected inthe nucleus shortly after IL-1β stimulation for 30 min. The levels ofUSF-2 remained unchanged after IL-1β stimulation. Fig. 3B shows thatpre-treatment with bavachin decreased IL-1β-induced nuclear trans-location of p65 and p50 and meanwhile, bavachin treatment resultedin retention of more p65 and p50 in the cytosol of IL-1β-treated cells.

The inhibition of nuclear translocation of NF-κB by bavachinsuggests that this drug may have effects on the NF-κB-associatedinhibitory protein, IκBα, which retains NF-κB in an inactive status inthe cytosol. We treated CHON-002 cells with 5 ng/ml IL-1β for theindicated time points and the cytosolic IκBα was determined byWestern blot. IL-1β stimulation induced IκBα degradation as early as10 min after treatment (Fig. 3C). In the presence of bavachin, the IL-1β-induced degradation of IκBαwas prevented that resulted in partialbut significant recovery of the cytosolic levels of IκBα (Fig. 3D).

Because IκBα degradation was initiated through phosphorylationof 32 and 36 serine residues by IKKs, we further determine whetherbavachin has any effect on IKK activities. Chondrocytes were pre-treated with bavachin for 24 h and then treated with IL-1β and thewhole cell lysates were prepared for Western blotting to measure the

levels of phosphorylated forms of IKK. As shown in Fig. 3E, the IL-1β-induced IKKα/β phohsphorylation was greatly reduced by bavachintreatment. The total levels of both IKKα and IKKβwere not affected bybavachin treatment.

3.4. Bavachin inhibited chemokine production from IL-1β-stimulatedCHON-002 cells and freshly prepared human chondrocytes

After translocation from the cytosol to the nucleus, NF-κB binds toDNA and activates many cytokine and chemokine genes. To examinethe possible physiological significance of IKK-IκBα-NF-κB suppressionin cellular function, we determine whether bavachin could inhibit IL-1β-induced chemokine production. We show that bavachin signifi-cantly inhibited IL-1β-induced production of chemokines, includingRANTES, MCP-2, MIP1-α, and MIP1-β in CHON-002 cells in a dose-dependent manner (Fig. 4A). Similar decrease of chemokine produc-tion by bavachin was also demonstrated in chondrocytes freshlyprepared from knee joints of osteoarthritis patients receiving surgery(Fig. 4B). The consequence of decreased chemokine production bybavachin was further addressed with chemotaxis assays. The culture

Fig. 2. Bavachin inhibited NF-κB DNA-binding and transcriptional activity in a dose-dependent manner. CHON-002 cells at 1×106/ml were pretreated for 24 h with differentconcentrations of bavachin (1–20 μM) and then were stimulated with 5 ng/ml IL-1β for 2 h. The nuclear extracts were prepared for the measurement of NF-κB (A) or AP-1 (B) DNA-binding activity by EMSA. In (C), CHON-002 cells at 1×106/ml weremixed together with pNF-κB-luciferase/pTK-Renilla-luciferase reporter plasmids and the transfection reagents asdescribed inMaterials andmethods. Eight h after transfection, the cells were equally divided and pretreated or not with bavachin at different dosages for 24 h. After stimulation with5 ng/ml IL-1β for another 24 h, cells were collected and the total cell lysates were analyzed for luciferase activities. The representative data out of at least three independentexperiments are shown. *Pb0.05 versus IL-1β-treated only by paired Student's t-test.

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supernatants from IL-1β-stimulated cells in the presence or in theabsence of bavachin were collected and loaded onto the lowerchambers of the transwell cassette. The upper chambers were loadedwith 1×105 THP-1 cells. Cell migration from upper to lower chamberswas calculated by flow cytometry. By chemotaxis assays, we furthershow that under IL-1β stimulation, the culture supernatants frombavachin-treated cells compared to that from cells treated in theabsence of bavachin were less chemoattractive to THP-1 cellmigration (Fig. 5).

4. Discussion

Phytoestrogens are shown to reduce bone loss in women (Becket al., 2005). Other effects such as the reduction of cardiovascular risk,positive effects on cartilage metabolism, and anti-tumorigenicproperties have also been reported with phytoestrogens (Messinaet al., 1994; Ullmann et al., 2005). With regard to these properties, it isvery likely that phytoestrogens may preserve anti-inflammatoryeffects. The present study was undertaken to examine and identifypotential phytoestrogens preserving inhibitory effects on IL-1β-induced NF-κB signaling in chondrocytes. Among the investigatedphytoestrogens, we observe that bavachin, at high concentrations(10–20 μM), was capable of decreasing IL-1β-induced activation ofNF-κB signaling pathways. We demonstrate that bavachin inhibitedIL-1β-induced DNA-binding and nuclear translocation of p65 and p50,degradation of IκBα, IKK activity as well as NF-κB transcriptionalactivity. In addition, bavachin decreased IL-1β-induced production ofchemokines, including RANTES, MCP-2, MIP-1α, andMIP-1β. Through

reducing IL-1β-induced chemokine production, bavachin was alsocapable of indirectly inhibiting inflammatory cell migration.

Human articular chondrocytes have been reported to expressestrogen receptors alpha and beta (Ushiyama et al., 1999), andpresumably the chondrocyte cell line, CHON-002, derived from thelong bone of female fetusmay also express estrogen receptors. Estrogentreatment brings many beneficial effects in cartilage metabolism,including the promotion of chondrocyte proliferation and maturation,the inhibition of pro-inflammatory cytokines andMMPsproduction andthe protection against damage by reactive oxygen species (Tanko et al.,2008). Given the report from Women's Health Initiative suggestingpotential negative risk/benefit ratio for estrogen replacement therapy inmenopausal women, it is difficult to consider estrogen replacementtherapy as an important therapeutic option against progression ofosteoarthritis pathogenesis (Harman et al., 2004). Being somewhatdifferent from estrogen, phytoestrogens have both agonist andantagonist actions, depending on several factors such as the affinity toestrogen receptors, the ability of the ligand–receptor complex to attractspecific co-activator or co-repressor proteins and the concentration ofcirculating endogenous estrogens (Setchell, 2001). By antagonisticeffects, phytoestrogen soybeans can inhibit the proliferation of breastcancer cells (Barnes et al., 1990). Only limited kinds of phytoestrogensare reported to preserve therapeutic roles in cartilage destruction andmost of them are flavonoids. For example, daizdein and genisteinenhance insulin effects that stimulate glycosaminoglycan and proteo-glycan synthesis in female bovinearticular chondrocytes (Claassen et al.,2008). Soy isoflavones are capable of alleviating symptoms ofosteoarthritis patients (Arjmandi et al., 2004). Although there is

Fig. 3. Bavachin inhibited IKK-IκBα-NF-κB signaling pathway. CHON-002 cells at 1×106/ml were treated with 5 ng/ml IL-1β for 30, 60 or 90 min. The cells were collected and nuclearextracts were probed for expression of p65, p50 and USF-2 (as a control) by Western blot (A). In (B), CHON-002 cells at 1×106/ml were pretreated for 24 h with differentconcentrations of bavachin (1–20 μM) and were then stimulated with 5 ng/ml IL-1β for 30 min. The expressions of p65 and p50 in both cytoplasmic and nuclear extracts weredetermined by Western blot. N: nucleus; C:cytosol. In (C), CHON-002 cells were treated with 5 ng/ml IL-1β for the indicated time points. Cytoplasmic extracts were probed for theexpression of IκBα by Western blot. In (D), after pre-treatment with bavachin at various dosages for 24 h, CHON-002 cells were stimulated with 5 ng/ml IL-1β for 10 min.Cytoplasmic extracts were probed for the expression of IκBα byWestern blot. In (E), CHON-002 cells were pretreated with bavachin at different dosages for 24 h and then stimulatedwith 5 ng/ml IL-1β for 10 min. Western blot analysis was performed using antibodies recognizing the phosphorylated form of both IKKα and IKKβ or using antibodies recognizingtotal IKKα, IKKβ or β-actin. p: phosphorylated. Data shown are representative of three independent experiments with similar results.

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evidence that genistein reduces cyclooxygenase-2 production in humanchondrocytes (Hooshmand et al., 2007), genistein compared tobavachin in the same examined concentration did not effectivelydecrease IL-1β-induced NF-κB DNA-binding activity (Fig. 1D).

The significance of down-regulating NF-κB signaling pathway bybavachin in the prevention of osteoarthritis pathogenesis was furtheraddressed by other reports examining the effects of 17beta-estradiol(E2). The ability of E2 to impairNF-κBp65 translocation into thenucleushas been demonstrated in IL-1β-treated chondrocytes (Richette et al.,2007) and in lipopolysaccharide-stimulated macrophages (Ghislettiet al., 2005). Interestingly, in mouse splenocytes, although the nucleartranslocationof p65, c-Rel, andRelB, is decreased, E2 up-regulatesNF-κBsignaling through selective induction of B cell lymphoma 3 (Bcl-3) (Daiet al., 2007). In human chondrocytes, the NF-κB inhibitor reduces IL-1β-induced MMP-3 and MMP-13 production (Liacini et al., 2002). Pulaiet al. (2005) show thatNF-κBplays critical roles infibronectin fragment-induced chondrocyte activation and production of cytokines andchemokines such as IL-6, IL-8, MCP-1, growth-related oncogen-α(GRO-α), GRO-β, and GRO-γ. These observations are in support of ourreport that increased levels of both p65 and p50 in response to IL-1βstimulation were clearly demonstrated in nuclear extracts of chron-

drocytes. Furthremore, the results demonstrated that bavachin de-creased the phosphorylation of IKKα/β and had no effect on total levelsof IKKα and IKKβ. This strongly indicates that the target of bavachinshould be the more upstream molecules that regulate the phosphory-lation and activation of IKKα/β. While the signaling pathway regulatingIKKα/β activation is beginning to be explored (Chariot, 2009; Kingeterand Schaefer, 2010),we currently have nodata to showwhichmoleculeis exactly targeted by bavachin.Nevertheless, the delineation of the IKK-IκBα-NF-κB signaling cascades targeted by bavachin does furtherexpand our knowledge regarding the molecular mechanisms of anti-inflammatory effects of this phytoestrogen as a potential cartilage-protective medication.

Chemokines are known to modulate cell adhesion, apoptosis andproliferation and play important roles in pathogenesis of osteoarthritis.Chemokines and chemokine receptors have been constitutivelyexpressed from chondrocytes and the effects are significantly enhancedin osteoarthritis joints (Appleton et al., 2007; Borzi et al., 2000).Chemokine GRO-α that is expressed at high level in osteoarthritis jointshas been shown to induce hypertrophic differentiation of chondrocytes(Merz et al., 2003). Yuan et al. (2001) also reported that MCP-1 andRANTES can inhibit proteoglycan synthesis and induce theproductionof

Fig. 4. Effects of bavachin on chemokine production from activated CHON-002 cells and freshly prepared chondrocytes from osteoarthritis patients. CHON-002 cells (A) or humanchondrocytes from osteoarthritis patients (B) at 1×106/ml were pretreated with various dosages of bavachin for 24 h and then stimulated with IL-1β for another 24 h. Thesupernatants were collected for the determination of RANTES, MCP-2, MIP1-α, and MIP1-β concentrations by ELISA. Background and positive controls represent bavachin-freeunstimulated and bavachin-free IL-1β-stimulated samples, respectively. The positive control was taken as 100% and the readings in bavachin-treated samples were calculated andrepresented as percentages of the IL-1β-stimulated positive control accordingly. The representative data out of at least three independent experiments are shown. *Pb0.05 versus IL-1β treated only by paired Student's t-test. ND: not detectable.

Fig. 5. Culture supernatants from IL-1β-stimulated cells in the presence or absence ofbavachin on THP-1 cell migration. CHON-002 cells were pretreated with variousdosages of bavachin for 24 h and then stimulated with IL-1β for another 24 h. Thesupernatants were individually collected and loaded onto the lower chambers of thetranswell cassette. The upper chambers were loaded with 1×105 THP-1 cells. Cellsmigrating from the upper chambers to the lower chambers were collected and countedwith flow cytometry as described in Materials and methods. The results out of threeindependent experiments are shown. *Pb0.05 versus IL-1β-stimulated only by pairedStudent's t-test.

187C.-C. Cheng et al. / European Journal of Pharmacology 636 (2010) 181–188

MMP-3. Thesedata reveal a catabolic pathwayprimed by chemokines inarticular cartilage and thus propose a therapeutic approach againstcartilage destruction by attenuating chemokine production in osteoar-thritis. In our study, we show that bavachin inhibited production ofchemokines in IL-1β-stimulated chondrocytes in a dose-dependentmanner. Because the expressionof these chemokines is regulatedbyNF-κB signaling pathway, our observations thus greatly enhance the clinicalprospective of bavachin in the treatment of osteoarthritis. In support ofour observation in different tissues cells, E2 has been shown to decreaseIL-1β-induced RANTES secretion, mRNA expression, and promoteractivity in keratinocytes (Kanda and Watanabe, 2003).

In summary, our study indicates that a purified phytoestrogen,bavachin, at high concentrations (10–20 μM), could reduce IL-1β-induced production of chemokines and activation of IKK-IκBα-NF-κBsignaling pathway in chondrocytes. The present study is thereforeunraveling new properties of a phytoestrogen bavachin as an anti-inflammatory compound. Furthermore, the results from this studysuggest phytoestrogens attractive candidates that can serve as potentialalternatives of disease-modifying anti-osteoarthritis medications.

188 C.-C. Cheng et al. / European Journal of Pharmacology 636 (2010) 181–188

Acknowledgments

This work is supported in part by grants from National ScienceCouncil (NSC-96-2314-B-016-015-MY3) and Tri-Service GeneralHospital (TSGH-C98-08-S02), Taiwan, R.O.C.

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