Melatonin inhibits the expression of the inducible isoform of nitric oxide synthase and exerts protective effects in a carrageenan-induced model of inflammation

Introduction: The role of melaiomn as an immunomodulaior is well established. The expression of the inducible isoform of nitric oxide synthase (iNOS) imponandy contnbutes to the pathophysiology of shock and inflammation. Here we first studied, in cultured murine macrophages, the role of melatonin in the regulation of the expression of iNOS. In addition, we investigated whether melatonin affects NO production and modulates the inflammatory response in response to carrageenan. Methods: The murine monocyte/macrophage cell lines J774.1 and RAW were grown in DMEM supplemented with 10 % fetal calf serum. Cells were treated with melatonin or vehicle 30 min prior to lipopolysacchande. At 24h. nitrite was measured in by the Griess reaction and viability with the MTT method. In addition. Northern md Western blotting and promoter analysis in cells transiently transfected with hicifenoeiNOS promoter constructs were performed as described (Szabö et al. Int. J. OiteoL 1997; 10: 1093). For the induction of carrageenan-induced pleurisy, rats were anesthetized and 0.2 ml saline alone or containing 1% X-camgeenan was injected into the pleura] cavity. The skin incision was closed and animals allowed to recover. At 4 h, the m1' were sacrificed. The chest was opened and the pleural cavity washed with 2 ml of saline solution with heparin and indomethacin. The exudale and washing were removed by aspiration and volume measured. Nitrite/nitrate were measured in the lavage fluids. Results: In macrophages stimulated with bacterial lipopolysacchande, melatonin at 1 nM-1 mM, decreased the production of nitrite. Melatonin reduced iNOS steady state mRNA levels and iNOS protein expression. Using iNOS promoter-luciferase constructs, we found that melatonin inhibits iNOS promoter activation. Inhibition of iNOS expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B. Based on these data, we concluded that melatonin inhibits NO production in rmmunostimulated macrophages by inhibiting the expression of iNOS. The inhibitory effect of melatonin on NO production were also apparent in carrageenaninduced models of inflammation. In a model of carrageenan-induced acute pleurisy, nitrite/nitrate concentrations significantly increased in the exudale after carrageenan challenge. Nitrite/nitrate levels were significantly reduced in dose-dependent manner in rats treated with melatonin (25 and SO mg/kg). Melatonin also reduced the degree of peritoneal exudation, and neutrophil migration in this model of inflammation. Conclusions: Thus, melatonin inhibits the expression of iNOS. This effect may importantly contribute to the anti-inflammatory properties of this pineal hormone.