The Role of Reactive Oxygen Species in the Expression of Tumor Necrosis Factor-α Cardiomyocytes Stimulated by Lipopolysaccharide in Neonatal Rat

Objective To investigate the role of reactive oxygen species (ROS) in the signal transduction mechanisms of lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) expression in neonatal rat cardiomyocytes and determine the downstream molecule in the signal pathway. Methods Cardiomyocytes were isolated and cultured from neonatal Sprague-Dawley(SD) rats. Expression of TNF-α mRNA and protein level was determined by RT-PCR and ELISA. ROS generation in cardiomyocytes was determined by peroxide specific probe 2',7'-dichlorofluorescin diacetate (DCF-DA). Activation of p38 MAPK was determined by Western blot analysis with phospho-specific antibody. Results (1)In the cardiomyocytes stimulated with LPS, expression of TNF-α mRNA was greater than that in untreated cells (0.29±0.07 vs 0.07±0.02, P0.01), which was attenuated by pretreatment with N-acetylcysteine (NAC), an antioxidant (0.13±0.03, P0.01). Similarly, compared with control group, TNF-α content in culture medium significantly increased in LPS group (83.6±14.5 vs 22.1±7.1 pg/mL, P0.01). Pretreatment with NAC inhibited the increase of TNF-α content induced by LPS (39.1±10.2 pg/mL, P0.01). (2)LPS increased the ROS generation in myocytes (83±15 vs 34±9, P0.01), which was blocked by pretreatment with NAC (19±4, P0.01). (3)p38 MAPK was activated in LPS-stimulated cardiomyocyte (0.34±0.07 vs 0.18±0.05, P0.01), which was inhibited by NAC (0.24±0.03, P0.01). Furthermore, Pretreatment with p38 MAPK specific inhibitor, SB203580, significantly decreased TNF-α content in culture medium induced by LPS (46.8±11.6 vs 85.5±22.4 pg/mL, P0.01). Conclusion ROS are, at least in part, involved in TNF-α expression by LPS-stimulated cardiomyocytes. p38 MAPK is probably an important downstream molecule in the signal pathway.