Effect of Phellodendron amurense in protecting human osteoarthritic cartilage and chondrocytes

Journal of Ethnopharmacology 134 (2011) 234–242

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Effect of Phellodendron amurense in protecting human osteoarthritic cartilage and chondrocytes Joo-Hee Kim a,1 , Jeong-Eun Huh b,1 , Yong-Hyeon Baek c , Jae-Dong Lee a , Do-Young Choi a , Dong-Suk Park c,∗ a

Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Republic of Korea Oriental Medicine Research Center for Bone & Joint Disease, East-West Neo Medical Center, Kyung Hee University, 149 Sangil-dong, Gangdong-gu, Seoul 134-727, Republic of Korea Department of Acupuncture & Moxibustion, College of Oriental Medicine, East-West Neo Medical Center, Kyung Hee University, 149 Sangil-dong, Gangdong-gu, Seoul 134-727, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 18 June 2010 Received in revised form 19 November 2010 Accepted 6 December 2010 Available online 21 December 2010 Keywords: Phellodendron amurense Osteoarthritis Cartilage protection Proteolytic enzymes Inflammatory cytokines Mitogen activated protein kinase

a b s t r a c t Ethnopharmacological relevance: Traditional medicine has been widely using Phellodendron amurense Rupr. (Rutaceae) to treat various inflammatory diseases including arthritis. Aim of the study: This study investigated the effects of Phellodendron amurense in protecting cartilage, including regulating the levels of aggrecanases, matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMP), proinflammatory cytokines and signaling of the mitogen activated protein kinase (MAPK) pathway in human osteoarticular cartilage and chondrocytes. Materials and methods: Explants from human osteoarthritis cartilage were cultured alone or in IL-1␣ for 7 days with or without Phellodendron amurense ethanol extract or celecoxib (40, 100, 200 ␮g/ml). The effect of Phellodendron amurense on matrix degradation induced by IL-1␣ in human articular cartilage was assessed by staining, and the quantities of sulfated glycosaminoglycan (GAG) and type II collagen were calculated from the culture media. The levels of aggrecanases, MMPs, TIMP, and PGE2 in the culture media were investigated using an enzyme-linked immunosorbent assay (ELISA). In addition, reverse transcription polymerase chain reaction (RT-PCR) evaluated the mRNA expression of aggrecanases, MMPs and TIMP. Furthermore, Western blot analysis was performed to identify the roles that Phellodendron amurense played in the ERK, JNK and p38 signaling pathways. Results: Phellodendron amurense showed no evident cytotoxicity on human articular cartilage. Phellodendron amurense significantly inhibited the IL-1␣-induced degradation of GAG and type II collagen from human osteoarticular cartilage in a concentration-dependent manner. Celecoxib did not significantly inhibit IL-1␣-induced release of GAG and only slightly reduced type II collagen. Phellodendron amurense also dose-dependently decreased the levels of aggrecanase-1 and -2, MMP-1, -3, and -13, whereas it increased TIMP-1 expression in human osteoarticular cartilage. Celecoxib only decreased MMP-1 and MMP-13 levels in human osteoarticular cartilage. In addition, Phellodendron amurense reduced the phosphorylation of extracellular signal regulated kinase (ERK)1/2, Jun NH2-terminal kinase (JNK) and activated phospho-p38 MAPK in a dose-dependent manner in human osteoarthritic chondrocytes. Conclusions: Phellodendron amurense inhibited osteoarticular cartilage and chondrocyte destruction by inhibiting proteoglycan release and type II collagen degradation, down-regulating aggrecanases, MMP activities and phospho-ERK1/2, JNK and p38 MAP kinase signaling, and up-regulating TIMP-1 activity. Therefore, our results suggest that Phellodendron amurense is a potential therapeutic agent to protect cartilage against OA progression. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +82 2 440 7702; fax: +82 2 440 7705. E-mail addresses: [email protected] (J.-H. Kim), [email protected] (J.-E. Huh), [email protected] (D.-S. Park). 1 These authors contributed equally. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.12.005

Osteoarthritis (OA), the most common joint disease, is a multifactorial degenerative disorder characterized by a progressive degradation of extracellular matrix (ECM) components, chondrocyte destruction, subchondral bone remodeling, and synovial inflammation (Felson, 2006). An imbalance in articular cartilage metabolism is considered to be important in OA pathogenesis

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Fig. 1. Representative HPLC chromatogram of standard berberine and the extract of Phellodendron amurense. Berberine was detected at round 44 min in this system.

and a key factor in its progression (Iannone and Lapadula, 2003). In healthy articular joints, cartilage homeostasis is maintained by balanced cytokine-mediated anabolic and catabolic processes (Dieppe et al., 2000; Poole, 2000). However, in OA the balance shifts toward catabolism, leading to cartilage destruction. The disease progression and structural changes show that aggrecan catabolites released into the synovial fluid, and the inflamed synovium over producing cytokines and growth factors, are central pathophysiological events in OA. The disease course is related to a number of complex pathways and mechanisms, among which is excessive production of proteolytic enzymes, such as aggrecanases and MMPs (Tortorella et al., 2001; Burrage et al., 2006). Both aggrecanases and MMPs degrade aggrecan, whereas MMPs degrade type II collagen (Tortorella et al., 2001; Burrage et al., 2006). Therefore, targeting these protease activities may stop the cartilage degradation in OA. The aggrecanases are 4 metalloproteinases (ADAMTS-1, -4, 5, and -9) that belong to the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. Aggrecanase-1 and aggrecanase-2 are the major proteases responsible for turning over articular cartilage (Powell et al., 2007). Aggrecanase-2 is constitutively expressed, while treating cartilage explants with IL-1 or TNF induces aggrecanase-1 (Tortorella et al., 2001). Several MMPs degrade ECM, including MMP-1, MMP-2, MMP3, MMP-9, and MMP-13 (Burrage et al., 2006). MMP-1 is the most abundant member of the MMP-family, which play an important role in breaking cartilage type II collagen. MMP-3 can cleave proteoglycans and gelatin as well as type II collagen (Ishikawa et al., 2005). MMP-13 is a major collagen cleaving enzyme (Neuhold et al., 2001). These enzymes degrade both type II collagen and PG in joints (Burrage et al., 2006). MMP activities are controlled by a TIMP, which inhibits all MMPs at a 1:1 stoichiometry by forming highaffinity complexes (Baker et al., 2002). In OA, cartilage destruction is closely related to chondrocyte dedifferentiation, which is modulated by multiple phosphorylation of several different mitogen activated protein kinase (MAPK) such as ERK-1/2, JNK, and p38. The ideal therapeutic agent for OA would not only reduce joint inflammation, but also reverse articular cartilage loss, restoring normal cartilage function (Hardy et al., 2002). Current pharmacologic treatments mainly aim to alleviate the OA symptoms, thus remain unsatisfactory (Laufers, 2004; Farkouh et al., 2007). Nonsteroidal anti-inflammatory drugs (NSAID), including celecoxib, have been used clinically for the past few years to treat arthritic disease. Despite their excellent efficacy and universal use in treat-

ing OA, their use is controversial due to the negative effects on cartilage and potential adverse effects (Akhund et al., 2003; Chang and Howden, 2004). Therefore, more suitable therapies that prevent cartilage degradation and possibly even regenerate cartilage with few side effects must be developed. Phellodendron amurense has been widely used to treat inflammatory diseases including arthritis. There is much evidence that Phellodendron amurense has anti-inflammatory (Cuellar et al., 2001; Li et al., 2003; Park et al., 2007), immuno-stimulatory (Park et al., 1999; Bao et al., 2006) and anti-tumor (Kumar et al., 2007) activites. There have been no reports on its ability to protect cartilage human osteoarthritic cartilage explants. We investigated the efficacy and mechanism of Phellodendron amurense in protecting cartilage in interleukin (IL)-1␣-treated human articular cartilage and chondrocytes. Our results suggest that Phellodendron amurense could possibly be used clinically to protect cartilage destruction in OA. 2. Materials and methods 2.1. Preparing and standardizing Phellodendron amurense extract The stem bark of Phellodendron amurense was purchased from a herbal supplier in Seoul (Korea), and the voucher specimen was deposited at the herbarium of the Pharmacy of Oriental Medicine of KyungHee Medical Center, KyungHee University (Seoul, Korea). The material was authenticated by professor Kim Nam Jae, Pharmacy of Oriental Medicine at KyungHee Medical Center, KyungHee University. One kilogram of Phellodendron amurense bark was extracted with 50% (v/v) ethanol–water at 60 ◦ C for 8 h. The extract was then filtered with 3 mm paper with a 10 ␮m pore size, and the ethanol was removed by vacuum rotary evaporation (Eyela, Japan). The concentrate was freeze-dried and yielded 12.5%. Phellodendron amurense was standardized based on the berberine content, determined using a Waters-600S controller HPLC instrument (Waters Corporation, MA, USA), equipped with a 626 pump, an in-Line degasser AF, a 717 plus auto-sampler and a Waters 996 photodiode array detector (PAD) used at room temperature. A linear gradient elution of solvent A (ultra-pure water with 0.1% formic acid, 2 mM ammonium acetate) and solvent B (acetonitrile) was applied with the following program. A: 0–20 min, 10–35%; B: 20–35 min, 35–50%; C: 35–45 min, 50–85%. Berberine was detected at a mean level of 2.61 ± 0.12% at 345 nm (Fig. 1).

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2.2. Human cartilage explant culture Human OA cartilage was obtained from the femoral chondyle and tibia plateau of 19 patients undergoing total knee arthroplasty at the KyungHee University Medical Center. The average patient age was 68 years and was evenly distributed between 5 males and 14 females. NSAID medication was stopped 7 days before surgery, thus previous medication use is not expected to interfere. Two orthopedists read sites from all regions of the knee joint under a microscope. Only cartilage (2400 ± 580 mg cartilage per person) that appeared to be full thickness with significant fibrillation was selected, so most joints appeared worse than the cartilage used here. Cartilage was collected according to the medical ethical regulations of the KyungHee University Medical Center. Cartilage slices were cut aseptically as thick as possible from the articular bone surface, cut into square pieces, weighed aseptically (range 25 ± 0.1 mg) and cultured individually in 48-well plates with 400 ␮l complete culture medium. The complete culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10 mM HEPES, penicillin (100 IU/ml), streptomycin (100 ␮g/ml), and 5% fetal bovine serum (FBS). After 24 h, cartilage medium was changed to basal culture medium (DMEM, supplemented with 10 mM HEPES, penicillin 100 IU/ml, streptomycin 100 ␮g/ml and 2% FBS). 2.3. Treating cartilage explants with Phellodendron amurense Experimental groups were divided as follows; IL-␣unstimulated control group, IL-␣-treated group, IL-␣-treated group with Phellodendron amurense (40, 100, 200 ␮g/ml), IL-␣treated group with celecoxib (40, 100, 200 ␮g/ml, CELEBREX® Pfizer Pharmaceuticals LLC, USA). Cartilage pieces were placed in 48-well plates and treated with 5 ng/ml human recombinant IL-1␣ (R&D Systems, MN, USA) in basal culture medium. After a 1 h pretreatment, Phellodendron amurense or celecoxib was added to the basal culture media and then incubated in a humidified 5% CO2 incubator at 37 ◦ C. Reagents were replaced every 3–4 days, and supernatants were harvested at 7, 14, and 21 days. Supernatants were stored at −20 ◦ C until assayed. 2.4. Culture of human osteoarthritis chondrocytes Chondrocytes were isolated from pooled femoral and tibial cartilage from individual OA patients by incubating with 1 mg/ml trypsin (Sigma, MI, USA) for 1 h followed by an overnight digestion in 0.5 mg/ml type II collagenase. The following morning, the isolated chondrocytes were washed with complete medium, counted, and plated at 1 × 106 cells in poly-(2-hydroxyethyl methacrylate)coated 60 mm dishes. Cells were then cultured at 37 ◦ C for 12 h in 5% CO2 incubator. 2.5. Measuring cell viability Chondrocytes were plated at 2 × 104 cells/well in 96-well plates containing 100 ␮l of medium. After 18 h, cells were treated with different concentrations (0–1000 ␮g/ml) of drug (Phellodendron amurense or celecoxib) in the presence or absence of IL-1␣. After incubating for 72 h at 37 ◦ C, 10 ␮l of BrdU was added to each well, and the samples were incubated another 6 h at 37 ◦ C. Cells were fixed, anti-BrdU-POD was added, and detected by the TMB substrate reaction. The reaction was quantified by an ELISA reader at 480–650 nm. 2.6. Glycosaminoglycan (GAG) degradation assay GAG levels in the culture medium were determined by the amount of polyanionic material reacting with 1,9-

Table 1 Primer design for quantitative RT-PCR analysis. mRNA Aggrecanase-1 Aggrecanase-2 TIMP-1 MMP-1 MMP-3 MMP-13 GAPDH

Primersa annealing Tm (cycle) 5 -GTCTGTGTC CAGGGCCGATGC-3 55 ◦ C (32) 5 -GCCGCCGAAGGATCTCCAGAA-3 5 -GCGGATGTGTGCAAGCTGACC-3 55 ◦ C (32) 5 -AGTAGCC CATGCCATGCAGGA-3 Fw: 5 -GCA ACT CCG ACC TTG TCA TC-3 58 ◦ C (32) Rv: 5 -AGC GTA GGT CTT GGT GAA GC-3 Fw: 5 -TCA GTT CGT CCT CAC TCC AG-3 58 ◦ C (32) Rv: 5 -TTG GTC CAC CTG TCA TCT TC-3 Fw: 5 -ATG GAC CTT CTT CAG CAA-3 58 ◦ C (32) Rv: 5 -TCA TTA TGT CAG CCT CTC-3 Fw: 5 -AGG AGC ATG GCG ACT TCT AC-3 58 ◦ C (32) Rv: 5 -TAA AAA CAG CTC CGC ATC AA-3 Fw: 5 -GCT CTC CAG AAC ATC ACT CCT GCC-3 58 ◦ C (30) Rv: 5 -CGT TGT CAT ACC AGG AAA TGA GCT T-3

a Fw, forward; Rv, reverse; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tm, temperature.

dimethylmethylene blue. 20 ␮l samples were mixed with 100 ␮l DMB reagents (48 mg/ml DMB, 40 mM glycine, 40 mM NaCl, 10 mM HCl, pH 3.0) for 30 min at room temperature, and quantified spectrophotometrically at 590 nm (Spectramax, Molecular Devices, Sunnyvale, CA, USA). All measurements were performed in quadruplicate. Quantification was performed using a standard curve of chondroitin 6-sulfate from shark cartilage (Sigma) in the range of 0–35 ␮g/ml. Culture supernatant also measured protein concentration by Bradford solution (Pierce, Rockford, IL, USA) and then we calculated ␮g/mg. 2.7. Type II collagen degradation assay Type II collagen levels in the culture medium were determined using the Sircol Type II collagen Assay Kit (Biocolor Ltd., Valley Business Center, Northern Ireland). 100 ␮l of samples were mixed with Sirius red dye containing sulfonic acid, which reacts specifically with the basic side chain groups of type II collagens, for 30 min at room temperature using a mechanical mixer. After centrifuging for 10 min at 12,000 rpm, the unbound dye was removed, and the dye bound to type II collagen was redissolved in 0.5 N NaOH. Absorbance was measured at 540 nm using ELISA reader (Spectramax, Molecular Devices, CA, USA). All measurements were performed in quadruplicate. Concentrations were calculated using a standard curve in the range of 0–200 ␮g/ml with standards provided by the manufacturer. 2.8. Gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR) Cartilage was snap frozen in liquid nitrogen, immediately homogenized with Trizol® reagent (Invitrogen Corporation, CA, USA), and centrifuged at 12,000 rpm for 10 min at 4 ◦ C. One microgram of total RNA was reverse transcribed for 60 min at 42 ◦ C and then 15 min at 72 ◦ C, using an RT-PCR system (TaKaRa Biotechnology, Seoul, Korea) that contained RT buffer, oligo (dT) 12-mer, 10 mM dNTP, 0.1 M dithiothreitol, reverse transcriptase, and RNase inhibitor. PCR using primers specific for each cDNA was carried out in a 20 ␮l PCR reaction (as supplied by TaKaRa, Korea), supplemented with 2.5 units of TaKaRa TaqTM 1.5 mM each dNTP, 1× PCR buffer, and 20 pmol of each primer. The aggrecanase-1, aggrecanase-2, MMP-1, MMP-3, MMP-13, and TIMP-1 primers used are described in Table 1. An equal volume of each PCR was analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining. Marker gene expression was normalized to GAPDH expression in each sample. Signal intensity was quantified with the Gel Doc EQ (BIO-RAD Laboratories, Milan, Italy).

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Fig. 2. Effect of Phellodendron amurense on the viability of chondrocytes. Chondrocytes were exposed to each well at 0.01, 0.1, 1, 10, 100, 1000 ␮g/ml of Phellodendron amurense or celecoxib. After incubation of 3 days, the cell viability was determined by BrdU assay. Data were obtained from three independent experiments and each datum was represented as mean ± SEM.

2.9. Aggrecanase activity assay Total aggrecanase level was measured by colorimetric assay (Biosource), according to the manufacturer’s instructions. Supernatants from human cartilage explants cultured for 7 days were incubated in 1% (w/v) BSA in PBS/Tween 20 (0.05%, v/v) for 2 h at 25 ◦ C on a 96-well plate containing a monoclonal antibody that recognizes KS chains. According to the manufacturer, other non-KS glycosaminoglycans (GAGs), including hyaluronic acid, chondroitin sulfate, and heparin sulfate do not impacted this assay. Fragments containing the ARGSVIL neoepitope were detected using biotinylated mAb OA-1 (PIERCE, IL, USA). Bound biotinylated mAb OA-1 was detected using 1 ␮g/ml streptavidin-HRP and TMB as substrate. Absorbance following acidification were measured in a microplate reader 450 nm. Calibration curves for ARGSVIL peptide standards, used to quantitate ARGSVIL peptide produced in hydrolytic reactions, were run in parallel.

peroxidase-labeled anti-mouse IgG and immuno-reactive proteins were visualized using enhanced chemiluminescence. 2.13. Statistical analysis Results are expressed as the mean ± SD. Statistical significance was determined using one way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. p values less than 0.05 were considered statistically significant.

2.10. Enzyme-linked immunosorbent assay MMP-1, MMP-3, MMP-13, and TIMP-1 levels were measured by human enzyme-linked immunosorbent assay (ELISA) kits (R&D System Inc., MN, USA), according to the manufacturer’s instructions. The conditioned media were collected at 21 days, centrifuged at 1500 rpm for 5 min to remove cell debris, and stored at −70 ◦ C until use. MMP-1, MMP-3, MMP-13, and TIMP-1 level were measured in each sample. 2.11. Measuring prostaglandin (PG) E2 PGE2 production was determined from the supernatant of cultured cartilage explants using assay kits (R&D System). The PGE2 assay was performed per the manufacturer’s instructions. 2.12. Western blot analysis Chondrocytes were lysed with cold lysis buffer (Invitrogen, Carlsbad, CA, USA) on ice. Total protein (10 ␮g/lane) was size-fractionated by 10% SDS-polyacrylamide gel (Invitrogen) electrophoresis under reducing conditions and transferred to Hybond-C nitrocellulose membrane (Amersham Biosciences, NJ, USA). After blocking with 5% skim milk, the membrane was incubated with anti- phospho-ERK-1/2, phospho-p38, phosphoJNK, ERK, p38, JNK (Cell Signaling, MA, USA), COX-1 and COX-2 (Santa Cruz Biotechnology, CA, USA) or non-immune mouse IgG (Sigma). The membrane was then incubated with horseradish

Fig. 3. Effect of Phellodendron amurense on GAG and type II collagen degradation in human cartilage explants culture. Cartilage was cultured in quadruplicate in 400 ␮l of medium only, medium with 5 ng/ml IL-1␣, or medium with 5 ng/ml IL-1␣ plus different concentrations of Phellodendron amurense or celecoxib. GAG and type II collagen degradation are shown as the cumulative release into the culture medium. Data were obtained from three independent experiments and each datum was represented as mean ± SEM. ### p < 0.001 compared with control group, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with IL-1␣ group.

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Fig. 4. Effect of Phellodendron amurense on the level of matrixproteinases in human cartilage explants culture. Cartilages were cultured in medium only, medium with 5 ng/ml IL-1␣, or medium with 5 ng/ml IL-1␣ plus different concentrations of Phellodendron amurense or celecoxib. The supernatant was harvested and total RNA was isolated from cartilage explants culture. (A) The mRNA expression of aggrecanase-1, aggrecanase-2, MMP-1, MMP-3, MMP-13, and TIMP-1 were measured by RT-PCR from human cartilage culture. (B) The cumulative level of aggrecanages, MMP-1, MMP-3, MMP-13 and TIMP-1 levels were analyzed with ELISA assay. Data were obtained from three independent experiments and each datum was represented as mean ± SEM. ### p < 0.001 compared with control group, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with IL-1␣ group.

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Fig. 4. (Continued ).

3. Results 3.1. Effect of Phellodendron amurense on chondrocyte viability We examined whether Phellodendron amurense affects viability of human OA chondrocytes. Phellodendron amurense treatment alone, or Phellodendron amurense plus IL-1␣ were not cytotoxic, whereas celecoxib alone reduced viability of human OA chondrocytes during 3 days of culture (Fig. 2).

3.2. Effect of Phellodendron amurense on GAG and type II collagen degradation We initially investigated whether Phellodendron amurense affects GAG and type II collagen degradation in human cartilage explants. Phellodendron amurense, ranging from 40 to 200 ␮g/ml, showed dose-dependent inhibition of GAG release ranging from 16% to 53%. IL-1␣ treatment inhibited degradation of type II collagen in dose-dependent manner, by 62% or more in human cartilage explants. In contrast, celecoxib did not significantly reduce GAG release compared to controls, and slightly inhibited collagen degradation at 200 ␮g/ml. The type II collagen release was significantly less in the Phellodendron amurense-treated group than the

celecoxib-treated group (Fig. 3). These results suggest that Phellodendron amurense effectively blocks IL-1␣-induced degradation of GAG and type II collagen from human osteoarthritis cartilage explants.

3.3. Effects of Phellodendron amurense on aggrecanases, MMPs and TIMP We examined whether Phellodendron amurense regulates IL-1␣-mediated expression of aggrecanase-1, aggrecanase-2, MMP-1, MMP-3, MMP-13 and TIMP-1 mRNA from human cartilage explants. In the IL-1␣-treated group, expression of aggrecanase-1, aggrecanase-2, MMP-1, MMP-3 and MMP-13 mRNA increased significantly while TIMP-1 messages decreased. However, in the Phellodendron amurense-treated group, aggrecanase-1, aggrecanase-2, MMP-1, MMP-3 and MMP-13 expression markedly decreased, whereas TIMP-1 expression increased compared to the IL-1␣-treated group in a dose-dependent manner. In the celecoxib-treated group, MMP-1 and MMP-13 expression decreased significantly. Celecoxib did not affect aggrecanase-1, aggrecanase-2 or TIMP-1 levels in IL-1␣-treated cartilage explants (Fig. 4A). We then tested the levels of aggrecanases, MMP-1, MMP3, MMP-13 and TIMP-1 in the IL-1␣-treated culture media with

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or without Phellodendron amurense treatment. In the Phellodendron amurense-treated group, aggrecanases, MMP-1, MMP-3 and MMP-13 levels decreased significantly in a dose-dependent manner compared to IL-1␣ treatment alone. TIMP-1 level increased significantly and dose-dependently compared to IL-1␣ treatment alone group (Fig. 4B). 3.4. Effects of Phellodendron amurense on proinflammatory mediators The PGE2 level decreased significantly following Phellodendron amurense treatment in a dose-dependent manner, compared to IL1␣ alone. Celecoxib also inhibited PGE2 production. The ability of Phellodendron amurense to down-regulate PGE2 release and COX-2 in cartilage explants was similar to celecoxib (Fig. 5A and B). 3.5. Phellodendron amurense inhibits IL-1˛-induced chondrocyte de-differentiation via MAPK signaling pathway To understand the molecular mechanism by which Phellodendron amurense inhibits IL-1␣-induced de-differentiation, we investigated any involvement of MAP kinase pathway by Western blotting. Phellodendron amurense dose dependently diminished ERK phosphorylation, phospho-JNK and phospho-p38 MAPK. Celecoxib also slightly suppressed pERK activation, but did not affect either pJNK or pp38 activity (Fig. 6). 4. Discussion Phellodendron amurense is an oriental medicinal herb that has been used to treat inflammatory conditions, most notably arthritis. Phellodendron amurense, cork tree, is a genus of deciduous trees in the family Rutaceae, and used in the oriental medicine to clear damp-heat, purge fire and expel pathogenetic heat. Several studies reported that Phellodendron amurense extracts prevented prostate tumor development (Kumar et al., 2007), inhibited prostatic contractility (Xu and Ventura, 2010) and acted on the urogenital system (Dvorkin and Song, 2002). A pilot study in OA patients also showed that oral administration of Phellodendron amurense decreased symptoms and inflammation of OA (Oben et al., 2009). It has been effective and safe during its long history of human use (Huh, 1999; Kim, 2000), however, its use still lacks scientific support (Marcus and Grollman, 2002; Tapsell et al., 2006). Therefore, this study sought to demonstrate that Phellodendron amurense can protect a human in vitro model from articular cartilage degeneration.

Fig. 5. Effects of Phellodendron amurense on the levels of proinflammatory mediators. (A) The release of PGE2 was determined by ELISA. ### p < 0.001; the IL-1␣-non-treated culture (IL-1 (−)) compared with IL-1␣-treated culture (IL-1 (+)). **p < 0.01 and ***p < 0.001; the IL-1 (+) control compared with Phellodendron amurense or celecoxib-treated group. (B) The protein expression of COX-1 and COX-2 was analyzed by western blot analysis. ␤-Actin was used as an internal control.

In human OA cartilage explants, Phellodendron amurense significantly prevented IL-1␣-mediated GAG release into the medium in a dose dependent manner (Fig. 3). GAG released into the medium was used as an index of PG degradation, the biomechanical consequences of which have been examined in previous studies (DiMicco et al., 2004; Patwari et al., 2007). In contrast, celecoxib did not significantly inhibit GAG degradation. Moreover, Phellodendron

Fig. 6. Effect of Phellodendron amurense on pERK, pJNK and pp38 MAPK proteins in IL-1␣-stimulated chondrocytes. The protein expression of pERK, pJNK and pp38 was analyzed by western blot analysis. ␤-Actin was used as an internal control.

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amurense markedly reduced type II collagen degradation in a dosedependent manner compared to IL-1␣-treated cultures (Fig. 3). These inhibitory effects of Phellodendron amurense were not due to cytotoxicity, because Phellodendron amurense did not affect the cell viability even at the 1000 ␮g/ml concentration. These results show the good safety of Phellodendron amurense in vitro, but the in vivo safety data needs to be further evaluated (Fig. 2). Both Phellodendron amurense and celecoxib inhibited PGE2 release in human OA cartilage explants (Fig. 5A). PGE2 , an inflammatory mediator, is responsible for cartilage and bone remodeling (Hardy et al., 2002). Thus, Phellodendron amurense inhibits the pathologic inflammatory molecules that destroy cartilage in OA. Both Phellodendron amurense and celecoxib reduced COX-2 expression (Fig. 5B). Many studies have shown that proinflammatory substances (COX-2, PGE2 ) destroy components of the cartilage extracellular matrix and induce proteoglycan loss (Panico et al., 2004; Faour et al., 2006). Our data show that the Phellodendron amurense and celecoxib suppress PGE2 production and inhibit COX-2 expression similarly. Cartilage protection seems to result controlling key components in OA pathogenesis, including down-regulating aggrecanases and MMPs and up-regulating TIMP. Aggrecanase-1 is especially common in joint fluid in rheumatoid arthritis and osteoarthritis. Chondrocyte express aggrecanase-1 in diseased cartilage, suggesting that it plays an important role in cartilage destruction in such joint diseases (Nagase and Kashiwagi, 2003). Recent papers reported that deleting both aggrecanase-1 and -2 in mice significantly protected against proteoglycan degradation comparable to deleting aggrecanase-2 alone, and decreased the severity of murine OA (Majumdar et al., 2007). Some studies have shown that type II collagen and PG release from cartilage can be prevented by inhibiting MMP transcription (Sabatini et al., 2005; Piecha et al., 2010). In OA, up-regulated MMPs are considered critical to degrading ECM. Therefore, MMPs and TIMP would be reasonable therapeutic targets to treat osteoarthritis. We demonstrated that Phellodendron amurense markedly and dose-dependently decreased aggrecanase-1, -2, MMP-1, -3, and -13 levels; while increasing the TIMP-1 level without cytotoxicity (Fig. 4A and B). These results suggest that Phellodendron amurense controlled cartilage loss by down-regulating aggrecanases and MMPs, and up-regulating TIMP-1 in IL-1␣-treated cartilage explants. In contrast, celecoxib only decreased MMP-1 and -13, having no effect aggrecanase-1, aggrecanase-2, MMP-3 or TIMP-1 (Fig. 4A and B). Chondrocyte dedifferentiation destroys the structural and biochemical homeostasis in OA, and is modulated by MAP kinases, such as ERK-1/-2, JNK, and p38 kinase. Some studies also have shown that p38, JNK, and ERK are crucial for MMP production and aggrecanases (Mengshol et al., 2002; Vincenti and Brinkerho, 2002; Sondergaard et al., 2010). Thus, we investigated whether Phellodendron amurense inhibits IL-1␣-induced chondrocyte dedifferentiation through MAP kinases. Phellodendron amurense suppressed chondrocyte dedifferentiation by decreasing ERK-1/-2, JNK and p38 MAP kinase phosphorylation, while celecoxib only inhibited ERK 1/-2 phosphorylation. Thus Phellodendron amurense suppressed the matrix proteinases such as MMP-1, MMP-3, MMP-13, aggrecanase-1, and aggrecanase-2 by down-regulating all 3 MAP kinases, which could make Phellodendron amurense more effective than celecoxib in preventing cartilage destruction. In conclusion, Phellodendron amurense has a therapeutic effect on OA in vitro by protecting cartilage through aggrecanases/MMPs/TIMP more than celecoxib. Phellodendron amurense also inhibited chondrocyte dedifferentiation by down-regulating ERK/JNK/p38 MAP kinase signaling, and was not cytotoxicity. Therefore, Phellodendron amurense is a great candidate to develop further OA treatments.

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Acknowledgement This work was supported by a grant from the KyungHee University in 2010 (KHU-20100135).

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