Autoantibody responses could be amplified and main tained on repeated stimulation
Resveratrol is a polyphenolic phytoalexin that demonstrates anti inflammatory, anti tumour, immunomodulatory, selleckchem IAP inhibitor 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 scientific assay products mainly through proteasome inhibition. Sig nalling pathways have been found to malfunction selleck in chondrocytes and synovial cells in OA and RA. Anti bodies raised against anti active caspase 3, MMP 9 and MMP 3 were purchased from R D Systems. Cyclooxygenase 2 antibody was obtained from Cayman Chemical. Antibodies against p65, pan IB, Bcl 2, Bcl xL and TNF receptor associated factor 1 were obtained from Santa Cruz Biotechnology. Antibodies against phospho specific IBand against anti phospho specific p65 were obtained from Cell Technology.
Anti IBkinase and anti IKK were obtained from Imgenex. Anti vascular endothelial growth factor antibody was pur chased from NeoMarkers. Monoclonal anti PARP antibodies were purchased from Becton Dickinson. Sox 9 antibody was purchased from Acris Antibodies GmbH. All antibodies were used at concentrations and dilutions rec ommended by the manufacturer. Growth medium and chemicals Growth medium containing 10% FCS, 25 gml ascorbic acid, 50 IUml streptomycin, 50 IUml penicillin, 2. 5 gml amphotericin B, essential amino acids and L glutamine was obtained from Seromed. Trypsinethylenediamine tetraacetic acid was purchased from Sigma. Epon was obtained from Plano. The alkaline phosphatase based APAAP kit was purchased from Dako. Resveratrol was purchased from Sigma. Curcumin was purchased from Indsaff. Resveratrol was prepared as a 100 mg ml solution in ethanol and then further diluted in cell culture medium. Curcumin was diluted in DMSO as a 5,000 M con centration and then further diluted in cell culture medium. IL 1 was obtained from Strathman Biotech GmbH. Peptide aldehydes and the specific proteasome inhibitor N Ac Leu Leu norleucinal were obtained from Boe hringer Mannheim. Chondrocyte isolation and culture Cartilage tissue samples from healthy femoral head articular cartilage obtained during joint replacement surgery for femoral neck fractures were used to isolate primary human articular chondrocytes. Cartilage slices were digested primarily with 1% pronase for 2 hours at 37 C and subsequently with 0. 2% collagenase for 4 hours at 37 C. 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. 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.