Figure 4

Gene expression and production of matrix metalloproteinase-13 in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG), specific inhibitors for mitogen-activated protein kinases and NF-κB on the gene expression of matrix metalloproteinase (MMP)-13 in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. Primary human chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated with AGE-BSA (600 μg/ml) for 8 hours. Expression of MMP-13 mRNA was normalized to GAPDH and compared with the levels present in control. Concentrations of specific inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB202190) and NF-κB (MG-132) used in these studies were 10 μM, 50 μM, 100 μM and 100 μM, respectively. Native BSA (600 μg/ml) was used as negative control. Results are representative (mean ± standard error of the mean) of duplicate experiments with chondrocytes obtained from five age-matched and sex-matched OA donors; data without a common letter differ, P < 0.01. (b), (c) Effect of EGCG on the production of MMP-13 in AGE-BSA-stimulated OA chondrocyte culture medium. Primary chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated with AGE-BSA (600 μg/ml) for 24 hours. MMP-13 production was analyzed in cell culture supernatant by (b) western blotting and (c) gelatin zymography. Equal volumes of culture supernatant were loaded on polyacrylamide gel. The MMP-13 positive control (EMD Chemicals) was also used. Band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software and are expressed in arbitrary optical density units. Data shown are cumulative of two experiments. OD values presented as mean ± standard deviation; data without a common letter differ, P < 0.05.