Inhibition of IL-1β-induced NF-κB activation and nuclear translocation by resveratrol and curcumin in primary chondrocytes using APAAP. (a) Human chondrocyte cultures either served as controls (panel A, not treated) or treated either with 10 ng/ml IL-1β 5, 15 and 30 minutes alone or pre-treated with 50 μM resveratrol and 50 μM curcumin for 4 hours and then co-treated with 10 ng/ml IL-1β for 5, 15 and 30 minutes, before immunolabelling with phospho-p65 antibodies. In control cells, anti-phospho-p65 labelling was restricted to the cytoplasm (panel A). Cells treated with IL-1β alone revealed nuclear translocation of phospho-p65 (panels B-D) that was inhibited by co-treatment with resveratrol and curcumin (panels E-G). Data shown are representative of three independent experiments. A-G: × 160, bar = 50 μm. (b) Panel I: western blot analysis with IL-1β-treated chondrocyte nuclear extracts. Serum-starved chondrocytes were pre-incubated with 50 μM resveratrol and 50 μM curcumin for 5, 10, 20, 30, 40 and 50 minutes, co-treated with 10 ng/ml IL-1β for 30 minutes, and then probed for phospho-p65 by western blot analysis using antibodies to phospho-specific p65 and poly(ADP-Ribose) polymerase (PARP) (control). Resveratrol and curcumin pre-treatment inhibited IL-1β-induced NF-κB activation in a time-dependent manner. NF-κB nuclear translocation was inhibited completely after 50 minutes of pre-treatment with resveratrol and curcumin. Panel II: serum-starved human chondrocytes were pre-incubated with resveratrol and curcumin at various concentrations (5 μM, 10 μM, 20 μM, 30 μM, 40 μM and 50 μM each) for 4 hours followed by 10 ng/ml IL-1β stimulation for 30 minutes. The nuclear extracts (500 ng protein per lane) were probed for phospho-p65 by western blot analysis using antibodies to phospho-specific p65 and PARP (control). A concentration-dependent inhibition of NF-κB nuclear translocation was observed. At a concentration of 40 μM resveratrol and 40 μM curcumin, NF-κB nuclear translocation was completely inhibited. The inhibition of NF-κB nuclear translocation by resveratrol and curcumin is therefore concentration as well as time dependent. Synthesis of PARP remained unaffected in nuclear extracts.