[Effect of hydrogen sulfide on H2O2-stimulated primary neonatal rat cardiomyocytes].

ABSTRACT

OBJECTIVE: To investigate the effects of hydrogen sulfide (H2S) on H2O2-stimulated primary neonatal rat cardiomyocytes and related mechanism. METHODS: Primary neonatal rat cardiomyocytes were treated with various concentrations of H2O2 (10, 100, 1000 µmol/L) for 24 h to establish the oxidative stress-induced cell injury model after 3 days' conventional culture. In addition, different concentrations of NaHS (1, 10, 100 µmol/L) were added to cardiomyocytes in the absence and presence of 100 µmol/L H2O2 for 24 h. The viability of cardiomyocytes was measured by MTT assay. The SOD vitality was measured by xanthine oxidase method and MDA content was determined by thiobarbituric acid colorimetric method. LDH activity was measured by chemical colorimetric method. The percentage of apoptotic cells was assessed by flow cytometry (FCM). The mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 (Rh123) staining and photofluorography. The level of reactive oxygen species (ROS) in cardiomyocytes was measured by DCFH-DA staining and photofluorography. RESULTS: Cell viability and SOD vitality were significantly reduced while MDA content and LDH activity were significantly increased with increasing H2O2 concentrations. These effects could be partly reduced by cotreatment with H2O2 in a concentration-dependent manner (all P < 0.05). Compared with control group, the DCF fluorescence intensity significantly increased in the 100 µmol/L H2O2 group (P = 0.003), which could be attenuated by NaHS in a dose-dependent manner. Compared with control group, the MMP significantly decreased in the 100 µmol/L H2O2 group (P = 0.000), which could be partly reversed by cotreatment with NaHS in a dose-dependent manner. Moreover, H2O2 treatment also significantly reduced 100 µmol/L H2O2 induced apoptosis in a dose-dependent manner. CONCLUSION: H2S protects primary neonatal rat cardiomyocytes against H2O2-induced oxidative stress injury through inhibition of H2O2 induced overproduction of ROS, dissipation of MMP and apoptosis.