Figure 5
Effects of resveratrol and curcumin on IL-1β-induced NF-κB-dependent gene products in primary chondrocytes. (a) The effect of resveratrol/curcumin on IL-1β-induced NF-κB-dependent anti-apoptotic gene products in primary chondrocytes was studied. To determine whether resveratrol and curcumin treatment actively stimulates the production of anti-apoptotic gene products, primary chondrocyte cultures were either stimulated for 0, 12, 24, and 48 hours with 10 ng/ml IL-1β or pre-treated with resveratrol and curcumin (50/50 μM) followed by 0, 12, 24, and 48 hours stimulation with 10 ng/ml IL-1β. Equal amounts (500 ng protein per lane) of total proteins were separated by 10% SDS-PAGE and analysed by immunoblotting with anti-Bcl-2, anti-Bcl-xL and anti-TNF-α receptor-associated factor 1 (anti-TRAF1) antibodies. A time-dependent downregulation of the expression of Bcl-2, Bcl-xL and TRAF1 by IL-1β was observed. In contrast, pre-treatment with resveratrol and curcumin resulted in a time-dependent increase of these anti-apoptotic proteins. Synthesis of the housekeeping protein β-actin remained unaffected. (b) The effect of resveratrol/curcumin on IL-1β-induced NF-κB-dependent pro-apoptotic protein caspase-3 was also studied in primary chondrocytes. Whole cell lysates of primary chondrocyte cultures were either stimulated for 0, 12, 24, and 48 hours with 10 ng/ml IL-1β or pre-treated with resveratrol and curcumin (50/50 μM) followed by 0, 12, 24, and 48 hours of stimulation with 10 ng/ml IL-1β-, and evaluated with western blot analysis to examine the effect on the pro-apoptotic protein caspase-3. Equal amounts (500 ng protein per lane) of total proteins were separated by 12% SDS-PAGE and analysed by immunoblotting with an antibody against active caspase-3. Stimulation of the cultures with IL-1β resulted in a time-dependent activation of caspase-3. In contrast, combinational treatment of resveratrol and curcumin inhibited caspase-3 activation in a time-dependent manner. Synthesis of the housekeeping protein β-actin was not affected.