Figure 5

EGCG mediated inhibition of NF-κB and MAPKs in IL-1β stimulated human OA chondrocytes and regulation of IL-6, IL-8 and TNF-α. (a) Human OA chondrocytes were pretreated with EGCG (10 to 100 μM) for 2 h and stimulated with IL-1β for 24 h. NF-κBp65 was determined in cell lysate by highly sensitive and specific ELISA (Assay design). TNF-α-treated extract of HeLa cells (supplied with the kit) was used as a positive control. The assay is developed with a chemiluminescent substrate and the signal is detected using luminometer (Lumat LB 9507; Berthold Technologies). NF-κB p65 activity was expressed as relative light unit (RLU). (b) After pretreatment with EGCG (10 to 100 μM) for one hour, chondrocytes were stimulated with IL-1β for one half-hour, and then phosphorylation of JNK, ERK and p38-MAPK was determined by Western immunobloting. (c, d, and e) Effect of specific inhibitors for mitogen activated protein kinases and NF-κB on the gene expression of IL-6, IL-8 and TNF-α in IL-1β stimulated human OA chondrocytes. Primary chondrocytes were pretreated with specific inhibitors for 2 h and were stimulated with IL-1β for 6 h. Relative gene expression of IL-6, IL-8 and TNF-α normalized to GAPDH and compared with un-stimulated control, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of p38 (SB202190), JNK (SP600125), ERK (PD98059) and NF-κB (MG132) used in these studies were 100 μM, 10 μM, 50 μM and 100 μM, respectively. Value represents Mean ± SE of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. EGCG, epigallocatechin-3-gallate;IL-interleukin; ERK, extracellular signal-regulated kinases; JNK, cJun-N-termial Kinases; MAPKs, mitogen activated protein kinases; NF-κB, nuclear factor kappa-B; TNF, tumor necrosis factor.