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Journal of Ethnopharmacology 111 (2007) 213–218

A purified extract from Clematis mandshurica preventsstaurosporin-induced downregulation of 14-3-3and subsequent apoptosis on rat chondrocytes

Sung Won Lee a, Sun Mee Choi a, Yun Suk Chang b, Kyung Taek Kim b, Tae Hyun Kim c,Hwan Tae Park d, Bong Soo Park e, You Jeong Sohn a, Soo Kyung Park a,

Su Hyun Cho a, Won Tae Chung a, Young Hyun Yoo c,∗a Department of Rheumatology, Dong-A University College of Medicine and Medical Science Research Center,

3-1 Dongdaesin-Dong, Seo-Gu, Busan, South Koreab Department of Orthopaedics, Dong-A University College of Medicine and Medical Science Research Center,

3-1 Dongdaesin-Dong, Seo-Gu, Busan, South Koreac Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Medical Science Research Center,

3-1 Dongdaesin-Dong, Seo-Gu, Busan, South Koread Department of Physiology, Dong-A University College of Medicine and Medical Science Research Center,

3-1 Dongdaesin-Dong, Seo-Gu, Busan, South Koreae Department of Oral Anatomy and Cell Biology, School of Dentistry, Pusan National University, Busan 602-739, South Korea

Received 19 January 2006; received in revised form 11 November 2006; accepted 16 November 2006Available online 21 November 2006

bstract

bjective: To dissect the mechanism of the protection of staurosporin-induced apoptosis on rat chondrocytes by a purified extract from Clematisandshurica.esign: Primary cultured rat articular chondrocytes as well as RCJ3.1C.18 cells were incubated with 1 �M staurosporin and 300 �g/ml purified

xtract from Clematis mandshurica. Western blot assay, silencing 14-3-3 gene and immunoprecipitation were conducted.esults: Clematis mandshurica prevented staurosporin-induced downregulation of several antiapoptotic bcl-2 family proteins Bcl-xL and Bcl-, and staurosporin-induced upregulation of an apoptotic bcl-2 family protein Bax. Clematis mandshurica also prevented staurosporin-inducedownregulation of a premitochondrial antiapoptotic protein 14-3-3. It is noticeable that siRNA to 14-3-3 abolished the prevention of caspase-3

ctivation by Clematis mandshurica. Furthermore viability assay corroborated that silencing of 14-3-3 gene abolished this apoptosis protectionfficacy by Clematis mandshurica. Immunoprecipitation assay elucidated that Clematis mandshurica prevented the staurosporin-induced reductionf the interactions between 14-3-3 with phospho-ser112-Bad and Bcl-xL to phospho-ser155-Bad.onclusions: Clematis mandshurica prevents staurosporin-induced apoptosis of rat chondrocytes via 14-3-3.2006 Elsevier Ireland Ltd. All rights reserved.

a

eywords: Cartilage; Apoptosis; Clematis mandshurica; 14-3-3

. Introduction

Osteoarthritis (OA) is a multifactorial disease with highorbidity that is characterized by degradation of the matrix

∗ Corresponding author. Present Address: Dong-A University College ofedicine, 3-1 Dongdaesin-Dong, Seo-Gu, Busan 602-714, South Korea.

el.: +82 51 240 2926; fax: +82 51 241 3767.E-mail address: yhyoo@dau.ac.kr (Y.H. Yoo).

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378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2006.11.012

nd destruction of articular cartilage (Mankin et al., 1971;eiss, 1973). The chondrocyte in cartilage is responsible for

he production and maintenance of the extracellular matrix.emarkable changes in articular cartilage include profound loss

n tissue cellularity and deleterious change of the extracellu-ar matrix in OA (Vignon et al., 1976; Mitrovic et al., 1983).

ecause the chondrocyte is the only type of cell that is pre-

ented in mature cartilage and is responsible for repairing theamaged tissue, reduced cartilage cellularity is considered tolay a critical role in the development of OA (Vignon et al.,

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976; Gardner, 1994). Recent studies showed that several differ-nt mechanisms may regulate apoptosis of chondrocytes (Blancot al., 1995; Hashimoto et al., 1997; Amin and Abramson, 1998;ashimoto et al., 1998; Lee et al., 2004). This indicates that

poptosis in chondrocytes is important to the pathogenesis of OAnd mechanism of cell death could be targeted for new treatmenttrategies.

In the treatment of OA, currently used drugs such asimple analgesics and non-steroidal anti-inflammatory drugsNSAIDs) are not curative and just only relieve the clinicaligns and symptoms of the disease rather than the underly-ng causes. Although these drugs generally decrease pain andtiffness, and improve function, beneficial effect to the under-ying cartilage and bone changes has been rarely demonstrated.owever, inhibiting the induction of apoptosis in chondrocytesay be a more fundamental therapeutic strategy to prevent

he destruction of articular cartilage. SKI 306X is a purifiedxtract from the mixture of three oriental herbs, i.e. Clema-is mandshurica, Trichosanthes kirilowii and Prunella vulgaris,hat have been widely used for the treatment of arthritis andther inflammatory diseases such as lymphadenitis in Far EastPark et al., 1995; Kim and Jeong, 1996). SKI 306X is knowno have cartilage protective, analgesic, and anti-inflammatoryfficacies (Jung et al., 2001; Choi et al., 2002), and our pre-ious study elucidated that SKI 306X had pharmacologicalffects in preventing apoptosis of cartilage and such activ-ty was mainly caused by Clematis mandshurica (Lee et al.,005).

We undertook this study to dissect the mechanism underly-ng this protection by Clematis mandshurica. Our data showhat this apoptosis preventing activity of Clematis mandshurican the chondrocyte cell line RCJ3.1C.18 and primary culturedat articular chondrocytes is mediated via the premitochondrialntiapoptotic factor 14-3-3.

. Materials and methods

.1. Reagents

The following reagents were obtained commercially: Mouseolyclonal anti-human Bad and phospho-specific Bad (ser112nd ser136) antibodies were from Oncogene (Cambridge, MA).ouse polyclonal anti-human phospho-specific Bad (ser155)as obtained from Cell Signaling Technology (Beverly, MA).abbit polyclonal anti-human Bax, Bcl-2, Bcl-xL, 14-3-3�nd caspase-3 antibodies were from Santa Cruz Biotechnol-gy (Santa Cruz, CA). Rabbit polyclonal anti-active caspase-3as obtained from BD Bioscience (Palo Alto, CA); Dul-ecco’s modified Eagle’s medium (DMEM) and fetal bovineerum (FBS) were from Gibco BRL (Gaithersburg, MD);imethylsulfoxide (DMSO), RNase A, proteinase K, apro-

inin, leupeptin, phenylmethylsulfonyl fluroride, collagenasend propidium iodide were from Sigma (St. Louis, MO); stau-

osproin was obtained from Calbiochem (San Diego, CA);nd SuperSignal West Pico enhanced chemiluminescence west-rn blotting detection reagent was from Pierce (Rockford,L).

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macology 111 (2007) 213–218

.2. Plant materials

The dried roots of Clematis mandshuria Rupr. (Ranuncu-aceae) and Trichosanthes kirilowii Maxim. (Cucurbitaceae) andhe dried flower and stem of Prunalla vulgaris L. (Labiatae) wereurchased from the Kyung-dong herbal market (Seoul, Korea),nd identified by Dr. Nam-Jae Kim (Faculty of East West Med-cal Research Institute, Kyunghee University, Korea). Voucherpecimens have been deposited in the Herbarium of East West

edical Research Institute, Kyunghee University, Korea andhe accession numbers for Clematis mandshurica, Trichosanthesirilowii, and Prunella vulgaris were EWMRI 97-011, EWMRI7-012, and EWMRI 97-013, respectively.

.3. Preparation of Clematis mandshurica, Trichosanthesirilowii, and Prunella vulgaris

Each component of SKI 306X was prepared by extractinghe dried roots of Clematis mandshurica and Trichosanthes kir-lowii, and the dried flower and stem of Prunella vulgaris with0% (v/v) ethanol in water for 4 h at 80 ◦C. After the extractedolution was filtered and evaporated in vacuo, the residue wasartitioned between n-butanol and water. The n-butanol layeras evaporated in vacuo and lyophilized for a complete removalf the residual solvent to yield dark-brown powder. Each com-onent was standardized by quantification of each referencetandard with HPLC system. The reference standards are oleano-ic acid for Clematis mandshurica, rosmarinic acid for Prunellaulgaris, and trans-cinnamic acid for Trichosanthes kirilowii.

.4. Cell culture

For the present study, we used not only a cell line but primaryultured cells. Since our previous study on the apoptosis protec-ion activity by Clematis mandshurica, which provides us theasis of the present study, was undertaken by using a rat chon-rogenic cell line RCJ3.1C.18, the same cell line is employed.urthermore, we also employed primary cultured chondrocytes

o examine its underlying mechanism in the cell culture systemore relevant to in vivo conditions.Articular chondrocytes were isolated from tissue fragments

n humeral and femoral heads on 21-day old Sprague–Dawleyats. Rats were used after our college ethics committee approvedhat our protocol fulfilled the guide of animal experience estab-ished by The Korean Academy of Medical Sciences. The cellsrom a single animal were obtained for each experiment. Thenely mined articular cartilage was digested under gentle shak-

ng for 2 h at 37 ◦C in DMEM containing 2 mg/ml collagenase.ells were then washed and seeded in culture flasks or 24-ell plate at a density of 104 cells/cm2. The primary culturesere established and maintained in DMEM containing 10%

etal calf serum and antibiotics (penicillin 100 U + streptomycin0 �g/ml). The medium was changed every 3 days. The first

assage cells were used in all experiments.

The immortalized rat chondrogenic cell line RCJ3.1C.18kindly provided by Dr. Jane Aubin, University of Toronto,ntario, Canada) was maintained at 37 ◦C with 5% CO2 in an

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ir atmosphere in MEM-� with 1.5 g/l NaHCO3 and 10−8 Mexamethasone supplemented with 10% FBS.

.5. Induction of apoptosis by staurosporin

Since staurosporin is a well-known example of chemicals thatnduce apoptosis as a result of an interaction with an intracellulararget, it was employed as an apoptotic stimulus. Stock solutionsf staurosporin (1 mM), made by dissolving the drug in DMSO,nd were kept frozen at −20 ◦C until use. Cells were treated forh with various concentration of staurosporin, incubated with

he fresh medium for additional 24 h and then harvested. Cellsere stained with trypan blue and then counted using a hema-

ytometer. Since the dose required for half-maximal inhibitionf viability was about 1 �M, this single time point and con-entration were utilized for further assessment of apoptosis. Toxamine the preventive effect of each component of SKI 306Xn staurosporin-induced apoptosis in chondrocytes, cells werereated with 1 �M staurosproin with or without 300 �g/ml C.ansdhurica, Trichosanthes kirilowii, or Prunella vulgaris.

.6. Trypan blue exclusion assay

To determined viability, cells were harvested, stained withrypan blue and then counted by a blind observer using a hema-ytometer.

.7. Western blot analysis

Cells (2 × 106) were washed twice with ice-cold PBS, resus-ended in 200 �l of ice-cold solubilizing buffer [300 mMaCl, 50 mM Tris–Cl (pH 7.6), 0.5% Triton X-100, 2 mMhenylmethylsulfonyl fluroride, 2 �l/ml aprotinin, and 2 �l/mleupeptin], and incubated 4 ◦C for 30 min. The lysates wereentrifuged at 14,000 rpm for 15 min at 4 ◦C, and sodiumodecyl sulfate (SDS) and sodium deoxycholic acid (0.2%nal concentration) was added. Protein concentration of cell

ysates were determined by the method of Bradford (Bio-ad protein assay), and 50 �g of proteins were loaded onto.5–12% SDS-polyacrylamide gels. The gels were transferred topolyvinylidene difluoride membrane (Amersham) and reactedith each antibody. Immunostaining with each antibody waserformed using SuperSignal West Pico enhanced chemilu-inescence substrate and detected with LAS-3000PLUS (Fujihoto Film Company, Kanagawa, Japan). To develop caspase-3nd active caspase-3 in a single blot, immunostaining was con-ucted with the mixture of anti-caspase and anti-active caspase-3ntibodies.

.8. si-RNA transfection

Cells grown to a confluency of 40–50% in six-well plates

ere transfected with 1 nM of siRNA. Twenty-one nucleotideNA with 3′-dTdT overhangs was synthesized by Dharma-on Research (Lafayette, CO) in the “ready-to-use” option.he AA-N19 mRNA targets were 14-3-3 target sequence

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macology 111 (2007) 213–218 215

5′-GAACTTCTTTGGCTGATAA-3′). Transfection of si-RNAas performed by using siPORT Amine (Ambion) and Opti-EM media according to manufacturer recommendations.

ight h after transfection, cells were incubated with the freshedia for 24 and treated with staurosporin as described above.

.9. Immunoprecipitation

Cell extracts were incubated with anti-14-3-3� antibody inxtraction buffer at 4 ◦C overnight. The immuno-complexesere precipitated with protein A-Sepharose beads (Sigma)

or 2 h, and washed five times with extraction buffer prioro boiling in SDS sample buffer. Immunoprecipitated pro-eins or aliquots containing 40 �g protein were separated onDS-polyacrylamide gels, and Western blot analysis to detecthophorylated Bad was performed as described.

.10. Statistical analysis

Four independent experiments were carried out. Statisticalesults were expressed as the mean ± the standard deviationf the means obtained from triplicates of each independentxperiment. The results of the experimental and control groupsere tested for statistical significance by the nonparametricruskall–Wallis test. In all cases, a p value was estimated less

han 0.05 was considered significant.

. Results

.1. Clematis mandshurica prevented staurosporin-inducededuction in the expression level of antiapoptotic factorsncluding 14-3-3

To dissect the mechanism underlying the prevention of apop-osis by Clematis mandshurica, the alteration of expressionevel of several representative apoptosis-related proteins in pri-

ary cultured chondrocytes were examined by Western blotting.estern blot assay demonstrated that staurosporin downregu-

ated the expression level of representative antiapoptotic bcl-2roteins Bcl-2 and Bcl-xL and upregulated an apoptotic bcl-2rotein Bax. Noticeably, Clematis mandshurica prevented theseffects of staurosporin on the expression level of these proteins.n the other hand, staurosporin did not significantly alter the

xpression level of another apoptotic bcl-2 protein Bad. Weext examined whether a premitochondrial antiapoptotic protein4-3-3 is involved in the prevention of apoptosis by Clema-is mandshurica. Western blot assay showed that staurosporinownregulated 14-3-3 and Clematis mandshurica preventedhis downregulation (Fig. 1). Although data are not shown,he similar data were also observed in chondrogenic cell lineCJ3.1C.18.

.2. Silencing 14-3-3 abolished the apoptosis protection

ffect of Clematis mandshurica

To explore that this apoptosis preventing activity of Clema-is mandshurica was mediated via 14-3-3, we specifically

216 S.W. Lee et al. / Journal of Ethnophar

Fig. 1. Clematis mandshurica prevented staurosporin-induced reduction in theexpression level of antiapoptotic factors including 14-3-3 in the primary culturedchondrocytes. C, control; S, staurosporin treatment; SC, combination treatmentwith staurosporin and Clematis mandshurica. Staurosporin downregulated anti-apoptotic proteins Bcl-2 and Bcl-xL, and upregulated an apoptotic bcl-2 proteinBax, and Clematis mandshurica prevented these effects of staurosporin. Stau-rosporin downregulated 14-3-3 and Clematis mandshurica prevented this effectof staurospoin. �-actin, internal control. The presented results are representativeof four independent experiments.

Fig. 2. Silencing 14-3-3 abolished the apoptosis protection effect of Clematismandshurica in chondrogenic cell line RCJ3.1C.18. C, control; S, staurosporintreatment; SC, combination treatment with staurosporin and Clematis mand-shurica; V, vehicle (siPORT Amine). SiRNA directed against 14-3-3 efficientlysilenced 14-3-3 gene and substantially reduced the expression level of 14-3-3,and silencing of 14-3-3 gene abolished the prevention of staurosporin-inducedprocaspase-3 degradation and its cleavage product formation by Clematis mand-shurica. As determined by trypan blue exclusion assay, silencing of 14-3-3abolished the prevention activity by Clematis mandshurica treatment (Fig. 2).*p < 0.05 (compared with SC). �-actin, internal control. Western blots presentedare representative of four independent experiments and results of viability assaywere expressed as mean ± S.D. of the means obtained from triplicates of eachindependent experiment.

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macology 111 (2007) 213–218

ecreased the expression of 14-3-3 by introducing 21-mer RNAligonucleotides into RCJ3.1C.18 cells by transfection. Smallnterfering RNA (siRNA) directed against 14-3-3 efficientlyilenced 14-3-3 gene and substantially reduced the expres-ion level of 14-3-3. It is noteworthy that silencing of 14-3-3bolished the prevention of staurosporin-induced procaspase-degradation and its cleavage product formation by Clematisandshurica. As determined by viability assay, silencing of 14--3 gene abolished this apoptosis protection efficacy of Clematisandshurica (Fig. 2). Although the viability percentages are not

he same, the similar alteration was also observed in primaryultured chondrocytes. Although the production of caspase-3leavage product was not well demonstrated in the primaryultured rat chondrocytes, silencing of 14-3-3 abolished the pre-ention of procaspase-3 degradation by Clematis mandshurica.s determined by viability assay showed that silencing of 14-3-also abolished this apoptosis protection efficacy of Clematisandshurica in primary cultured chondrocytes (Fig. 3).

.3. Clematis mandshurica treatment prevented thetaurosporin-induced reduction of the interactions between4-3-3 with phospho-ser112-Bad and BcL-xL with

hospho-ser155-Bad

Immunoprecipitaion data show that Clematis mandshuricarevented the staurosporin-induced reduction of the interac-

ig. 3. Silencing 14-3-3 abolished the apoptosis protection effect of Clematisandshurica in the primary cultured chondrocytes. C, control; S, staurosporin

reatment; SC, combination treatment with staurosporin and Clematis mand-hurica; V, vehicle (siPORT Amine). SiRNA directed against 14-3-3 efficientlyilenced 14-3-3 gene and substantially reduced the expression level of 14-3-3,nd silencing of 14-3-3 abolished the prevention of procaspase-3 degradation bylematis mandshurica. As determined by trypan blue exclusion assay, silencingf 14-3-3 abolished the prevention activity by Clematis mandshurica treatmentFig. 2). *p < 0.05 (compared with SC). �-actin, internal control. Western blotsresented are representative of four independent experiments and results of via-ility assay were expressed as the mean ± S.D. of the means obtained fromriplicates of each independent experiment.

S.W. Lee et al. / Journal of Ethnophar

Fig. 4. Clematis mandshurica treatment prevented the staurosporin-inducedreduction of the interactions between 14-3-3 with phospho-ser112-Bad and Bcl-xL with phospho-ser155-Bad in RCJ3.1C.18 cells. C, control; S, staurosporintreatment; SC, combination treatment with staurosporin and Clematis mand-shurica. Immunoprecipitaion data shows that Clematis mandshurica preventsthe staurosporin-induced reduction of the interactions between 14-3-3 withpcm

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hospho-Ser112-Bad and Bcl-xL with phospho-ser155-Bad. �-actin, internalontrol. The presented results are representative of four independent experi-ents.

ions between 14-3-3 with phospho-ser112-Bad and Bcl-xLith phospho-ser155-Bad in RCJ3.1C.18 cells (Fig. 4). The

imilar tendency was also observed in primary cultured chon-rocytes (Supplemental Fig. 1B). However, no interaction of4-3-3 with phospho-ser136-Bad was observed both in the con-rol and experimental cells (negative data not shown). To thisnd, we examined whether other two components of SKI 306, Trichosanthes kirilowii and Prunella vulgaris, modulate 14--3 in staurosporin-treated articular chondrocytes or not. Asur previous study indicated that Trichosanthes kirilowii andrunella vulgaris, on the contrary of Clematis mandshurica, didot prevent staurosporin-induced apoptosis (Lee et al., 2005),oth components, in the present study, altered neither celliability nor downregulation of 14-3-3 in primary culture artic-lar chondrocytes treated with staurosporin (Supplemental Fig.B).

. Discussion

Apoptosis of chondrocytes was initially reported in relationo development and growth, such as in the differentiation ofypertrophic chondrocytes and the ossification of the growthlate (Hatori et al., 1995; Bronckers et al., 1996). And, apop-osis was reported in human normal and OA cartilage (Blancot al., 1998; Kim et al., 2000) as well as in experimental mod-ls of OA (Hashimoto et al., 1998). It has also been reportedhat several stimuli, such as nitric oxide (Blanco et al., 1995;ashimoto et al., 1997, 1998; Amin and Abramson, 1998; Lee

t al., 2004), prostaglandin E2 (Miwa et al., 2000), Fas ligandFasL) (Hashimoto et al., 1997; Kuhn et al., 2000; Kuhn andotz, 2001), tumor necrosis factor and tumor necrosis factor-

elated apoptosis-inducing ligand (TRAIL) (Hochberg et al.,995a; Lee et al., 2004), induced apoptosis in chondrocytes,

hich indicated that those molecules might play a part in theathogenesis of OA. Thus, modulation of the mechanism medi-ted by such substances can be considered as a novel strategyor the treatment of OA.

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macology 111 (2007) 213–218 217

Although the standard treatment of OA, which is the onemong the most important causes of age-related pain and disabil-ty, includes analgesics, physical therapy, and surgery, analgesicsuch as paracetamol and non-steroidal anti-inflammatory drugsNSAIDs) represent the mainstay of treatment in medical man-gement of OA (Hochberg et al., 1995b). Recent reports showedhat NSAIDs nimesulide and ibuprofen, in addition to their anti-nflammatory and analgesic benefits, may also have a protectiveffect in OA through the inhibition of apoptosis in chondrocytesMukherjee et al., 2001). Accordingly, preventing apotosis inrticular chondrocytes appears to be a promising therapeutictrategy.

In this context, we have been studying the apoptosis prevent-ng efficacy of SKI 306X which is a solvent extract from theombination of three oriental herbal medicines, Clematis mand-hurica, Trichosanthes kirilowii and Prunella vulgaris. Eachomponent of SKI 306X has long been used as folk medicinen Korea in the treatment of some inflammatory diseases (Parkt al., 1995; Kim and Jeong, 1996; Jung et al., 2001). A clini-al study on patients with OA revealed that SKI 306X showedunctional improvements of the affected knee joint (Jung et al.,001). Also, SKI 306X inhibited proteoglycan degradation inartilage explant culture, and its prophylactic administration sig-ificantly protected the knee joint of rabbit from OA-like changen collagenase-induced experimental OA model (Choi et al.,002). Subsequently, we elucidated that only Clematis mand-hurica, among three components of 306X, showed apoptosisreventing efficacy, indicating that pharmacological efficacy ofKI 306X protecting OA in part results from the inhibition ofpoptosis in chondrocytes by Clematis mandshurica (Lee et al.,005).

The 14-3-3 proteins are a family of ubiquitously expressed,imeric proteins, consisting of seven known mammalian iso-orms (Fu et al., 2000). These proteins are multifunctionalhosphoserine binding molecules, and an emerging role for 14--3 is as an effector of prosurvival signaling (Fu et al., 2000).ecent studies suggested that this surviving effect is mediatedy the interaction of numerous proteins such as Bad (Zha etl., 1996). Bad is a member of the BH3-only subfamily of thecl-2 apoptosis-regulating proteins, which is regulated exten-ively by phosphorylation on serine. At least three sites on Badan be phosphorylated in vivo, including Ser112, Ser136, ander155 (Zha et al., 1996; Datta et al., 2000; Lizcano et al., 2000;hou et al., 2000). When Ser112 and Ser136 of Bad becomehosphorylated, Bad is found in the cytosol, bound to 14-3-3ather than Bcl-2 or Bcl-xL (Zha et al., 1996). On the otherand, phosphorylation of Ser155 disrupts the interaction of Bado Bcl-xL, which cause translocation of Bad from mitochondriao cytosol, resulting in the binding of Bad to 14-3-3 (Datta et al.,000). Since Bad binding to 14-3-3 does not promote apoptosis,hosphorylation of Bad is critical for cellular survival.

In conclusion, this study indicates that 14-3-3 is involvedn apoptosis of chondrocytes. This study also indicates that

poptosis prevention efficacy by Clematis mandshurica inhondrocytes is mediated via 14-3-3. Furthermore, this studyndicates that Clematis mandshurica prevented the decrease inhe interactions of 14-3-3 to phospho-ser112-Bad and Bcl-xL to

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binding to 14-3-3 BCL-X(L). Cell 87, 619–628.

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hospho-ser155-Bad. A better understanding of the mechanismnderlying inhibition of apoptosis in OA chondrocytes mightead to the development of a new therapeutic strategy for OA.

cknowledgements

This work was supported by grant no. R01-2004-000-10002-from the Korea Science & Engineering Foundation. Authors

hanks to Dr. Nam-Jae Kim (East West Medical Research Insti-ute, Kyunghee University, Korea) for helps on identification ofhe herbs.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.jep.2006.11.012.

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