Resveratrol is a polyphenolic phytoalexin that demonstrates anti inflammatory, anti tumour, immunomodulatory, scientific assays cardioprotective, anti oxidative and chem opreventive capabilities. We recently reported that resveratrol can inhibit IL 1 induced apoptosis in chondro cytes through downregulation of NFB regulated anti apop totic gene kinase inhibitor Carfilzomib products mainly through proteasome inhibition. Sig nalling pathways have been found to malfunction click here in chondrocytes and synovial cells in OA and RA. Cartilage samples were derived from human patients with full informed consent and local eth ics committee approval. Experimental design Chondrocyte monolayer cultures were washed three times with serum starved medium and incubated for 1 hour with serum starved medium. Serum starved human articular chondrocytes were either left untreated, treated with 10 ngml IL 1 alone for the indicated time periods, or pre treated with 50 M resveratrol, 50 M curcumin or 50 M res veratrol and 50 M curcumin for 4 hours followed by co treat ment with 10 ngml IL 1 and 50 M resveratrol, or 50 M curcumin or 50 M resveratrol and 50 M curcumin for 24 hours or for the indicated time periods. Cell viability and proliferation assay The effects of resveratrolcurcumin on the cytotoxic effects of IL 1 were examined by the 3 2,5 diphenyltetrazolium bromide uptake method as previously described. Briefly, for the cell proliferation assay, 5,000 chondrocytes per well were cultured for 24 hours in a 96 well plate and then treated with 10 ngml IL 1, 50 M resveratrol, 50 M curcumin, 50 M resveratrol and 50 M curcumin, or pre treated with 50 M resveratrol, 50 M curcumin, or 50 M resveratrol and 50 M curcumin for 4 hours and then co treated with 10 ngml IL 1, or left untreated and evaluated after 24 hours at 37 C. For evaluation, the medium was removed and 100 l fresh medium and 10 l MTT solution were added to each well and incubated for 4 hours at 37 C5% car bon dioxide. Subsequently, 100 l MTT solubilization solution was added and the plates incubated until the cells were bleached. The transmission signal was determined at 570 nm using a microplate reader. A sample without cell loading was used as a baseline value. The assay was performed in triplicate and the results are provided as mean values with standard deviations from three independ ent experiments. Poly polymerase cleavage assay To determine the cleavage products of the DNA repair enzyme PARP, serum starved chondrocytes were cultured for 24 hours and then treated with 10 ngml IL 1, with 50 M res veratrol, 50 M curcumin, and 50 M resveratrol and 50 M curcumin, or pre treated with 50 M resveratrol, 50 M curcu min, and 50 M resveratrol and 50 M curcumin for 4 hours and then co treated with 10 ngml IL 1, or left untreated for 24 hours at 37 C. Whole cell extracts were prepared and lysed in lysis buffer.
Lysates were spun at 14,000 rpm for 10 minutes to remove insoluble material, resolved by 7. 5% SDS PAGE, and probed with PARP anti bodies.