Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes

摘要

Objective: Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. Methods: Human cultured synoviocytes from OA patients were treated for 24h with interleukin 1-β (IL-1β), tumour necrosis factor α (TNF-α) or neither; for the last 6h, they were submitted to either normoxia or three periods of 1-hof hypoxia followed by 1-hof reoxygenation. NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2 - production, NOXsubunit expression and nitrosylation were also assessed. Results: iNOS expression and nitrite (but not peroxynitrite) production were significantly increased under H/R conditions when compared with to normoxia (P0.05). H/R conditions decreased O2 - production from ~0.20 to ~0.12nmolmin-1mg proteins-1 (P0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2 - production was restored at normoxic levels (0.21nmolmin-1mg of proteins-1). Conclusions: Our results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia.