Autoantibody responses could be amplified and main tained on repeated stimulation
Resveratrol is a polyphenolic phytoalexin that demonstrates anti inflammatory, anti tumour, immunomodulatory, IAP signaling pathway 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 Carfilzomib msds products mainly through proteasome inhibition. Sig nalling pathways have been found to malfunction Romidepsin structure in chondrocytes and synovial cells in OA and RA. Primary chondro cytes were cultured at a density of 200,000 cells in 60 mm petri dishes in monolayer culture for a period of 24 hours at 37 C with 5% carbon dioxide. Cartilage samples were derived from human patients with full informed consent and local eth ics committee approval. 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. NFB activation assay The effect of resveratrolcurcumin on the IL 1 induced nuclear translocation of p65 was examined by an immunocyto chemical method as described previ ously. Briefly, chondrocytes seeded on glass plates either were treated with 10 ngml IL 1 for 0, 5, 15 and 30 minutes alone, or were pre treated with resveratrol 50 M and curcu min 50 M for 4 hours and then co treated with 10 ngml IL 1 for 0, 5, 15 and 30 minutes. After incubation, cells were fixed for 10 minutes in ice cold methanol, washed twice in Tris buffered saline at ambient temperature and then pre incubated with normal serum for 10 minutes at ambient temperature.