When indicated, NOS inhibitors were added in the presence of adiponectin. 2 mM L NG monomethyl arginine citrate, a nonselective NOS inhi bitor, and 50 uM of L N6 lysine, a selective iNOS inhibitor. To ascertain the adiponectin related signaling path ways, OA chondrocytes were stimulated with adiponec tin in the presence of a kinase inhibitor. 10 uM SB202190 for p38 MAP kinase, 20 uM SP600125 for c Jun N terminal kinase, 50 uM U0126 for extracellular regulated kinase, 20 uM compound C for AMP acti vated protein kinase, 50 uM LY294002 for Akt, and 100 ugml SN50 for nuclear factor kappa B. No significant cytotoxicity was found for OA chondrocytes by the kinases or NOS inhibitors up to 24 hours of exposure. Measurement of NO and MMPsTIMP 1 levels in culture media The levels of total NO were measured by using a modi fied Griess reaction.
The concentrations of MMP 1, 3, and 13 and TIMP 1 in the conditioned p53 inhibitor media were analyzed by using commercial enzyme linked immunosorbent assay kits, which measured the pro MMP forms of MMP 1 and MMP 13 and the total forms for MMP 3. Western blotting iNOS expression in adiponectin stimulated OA chon drocytes was analyzed by immunoblotting by using anti iNOS and goat anti rabbit antibody. Adiponectin stimulated activation of AMPK and JNK was evaluated by using anti phospho AMPK and phos pho JNK antibodies. Reverse transcription polymerase chain reaction RNA expression levels of iNOS and MMPs were semi quantitatively determined by using the RT PCR with spe cific primer pairs Quantitative real time RT PCR was performed by using the ABI 7500 real time PCR machine.
The specific Taqman primers and probes hop over to here were purchased from Applied Biosystems. iNOS, MMP 1, MMP 3, MMP 13, and glyceraldehyde 3 phosphate dehydrogenase. The number fold difference in the expression of target mRNA was calculated by a comparative Ct method, normal ized to GAPDH. Measurement of collagenase cleaved type II collagen neoepitope To assess cartilage matrix degradation, the harvested OA cartilage tissue was cut into cubes of approximately 111 mm in size by using surgical blades. Cartilage pieces weighing a total of approximately 200 mg were placed into each well of a 24 well tissue plate with 1 mlwell of DMEM supplemented with 10% FBS. After 2 to 3 days, the cartilage explants were stimulated with FBS free DMEM including adiponectin or interleukin 1b for 8 days. During the treatment, the conditioned medium was harvested and replaced every 4 days. The concentrations of collage nase cleaved type II collagen product were measured in the harvested media by using a competitive immunoassay kit on days 4 and 8 after adiponectin treatment.