SW1353 cells (human chondrosarcoma cell line) purchased from the American type culture collection click here (Manassas, VA, USA) were cultured and treated with IL-1β according to previously described procedures . In brief, the cells were maintained in DMEM with 10% FBS, glutamine, and penicillin/streptomycin. To induce MMP-13, IL-1β (10 ng/mL) with/without test compounds was added to the cells in serum-free DMEM for 24 h. MMP-13 released in the media was examined by
Western blotting analysis using anti-MMP-13 antibody. All test compounds were initially dissolved in dimethyl sulfoxide (DMSO) and diluted with serum-free DMEM to adjust the final DMSO concentration to 0.1% (v/v). Cell viability was checked using MTT bioassay . No effect on cell viability or the MMP-13 expression level was observed by the treatment of 0.1% DMSO. Using total cellular lysate, expression and phosphorylation of MAPKs and STAT-1/-2 were examined. Total cellular protein was extracted with Pro-Prep solution (iNtRON Biotechnology, Kyungki-Do, Korea) containing 1mM phenylmethylsulfonyl fluoride (PMSF), 1mM sodium orthovanadate, and 1mM sodium fluoride. Expression of nuclear transcription factor-κB (NF-κB) p65, c-Jun, and c-Fos was identified in nuclear fractions. For an extraction of nuclear proteins, cells were resuspended in 400 μL of buffer
A (10mM HEPES, pH 7.9, 10mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) selleck kinase inhibitor and incubated on ice for 10 min. After 25 μL of 10% NP-40 was added, cells were vortexed for 10 sec and centrifuged at 2,500 g for 2 min. The nuclear pellet was vigorously vortexed in buffer B (20mM HEPES, pH 7.9, 0.4M NaCl, 1mM EDTA, 1mM DTT, 1mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin) and centrifuged at 16,000 g for 10 min. BCA protein assay (Pierce, IL, USA) was used to determine protein concentration in the nuclear fraction. Proteins were separated, blotted, and visualized as described
either above. According to the previously described procedures , articular cartilages were excised from the femoral condyles of rabbit knee and incubated in DMEM containing 5% FBS for 1–2 days. In addition, approximately 30 mg cartilage fragments per well were incubated in DMEM containing 1% FBS in 400 μL/well. Cartilages were treated with 10 ng/mL of human IL-1α (Sigma–Aldrich) in the presence or absence of test compounds for 3 days. The amounts of released GAG in the supernatant were measured with a Blyscan sulfated GAG assay kit (Biocolor, Carrickfergus, County Antrim, UK) based on dimethylmethylene blue assay, according to the manufacturer’s protocol. Experimental values are represented as arithmetic mean ± standard deviation. Statistical analysis was evaluated using one-way analysis of variance followed by Dunnett’s analysis (IBM SPSS Statistics, Version 21, IBM Korea). A p < 0.05 was considered significantly different.