Intermedin Protects H9c2Cardiomyocytes Against Hypoxia-reoxygenation-induced Oxidative Stress

Objective After transfection of IMD plasmid, cultured H9c2cardiomyocytes, then made hypoxia/reoxygenation (H/R) model. To prove the protective effects of IMD on the injury of oxidative stress, lay foundation for the research on the effects and molecular biology mechanism of IMD gene on the cardiovascular disease.Methods Cultured H9c2cell line of cardiomyocytes was randomly divided into4groups:1) normal control group:cultured H9c2cardiomyocytes with DMEM for48h;2) hypoxia/reoxygenation (H/R)group:H9c2cardiomyocytes cultured for48h were exposed to5mmol/l Na2S2O4for2h of hypoxia, followed by1h of reoxygenation;3) hypoxia/reoxygenation+blank plasmid group (blank plasmid group):after transfection of blank plasmid, cultured cell for48h, then made hypoxia/reoxygenation model;4) hypoxia/reoxygenation+IMD plasmid group (IMD group):after transfection of IMD plasmid, cultured cell for48h, then made hypoxia/reoxygenation model. In the end, the morphology of H9c2cardiomyocytes was observed by phase contramicroscope. In48hours after transfection of blank plasmid, the morphology of H9c2cardiomyocytes was observed by fluorescent microscope. Determine the best plasmid quantity by using the flow cytometry to detect the transfection efficiency. The content of LDH was detected by chemistry chromatometry. The content of MDA was detected by TBA colouration method. The activity of SOD was detected by xanthine oxidase method. The cardiomyocyte apoptosis was detected by flow cytometry.Results(1) H9c2cardiomyocytes exhibited adherent long spindle-shaped cells under phase contramicroscope.(2) Cardiomyocytes displayed green fluorescence under fluorescent microscopy, and exhibited adherent long spindle shaped cell.(3) The transfection efficiency of lug/ml plasmid is highest.(4) Compared with the control group, the levels of LDH and MDA in the H/R group was significantly increased, while the SOD activity was decreased (p<0.05). The transfection of IMD plasmid significantly reversed these changes. The indexes above improved obviously as compared to H/R group (P<0.05). There is no statistically significant between the blank plasmid group and the H/R group (p>0.05).(5) Compared with the control group, the cardiomyocyte apoptosis(medium and late stage)was increased in the H/R group(p<0.05).Compared with the H/R group, the apoptosis(medium and late stage) was decreased in the IMD group(p<0.05). There is no statistically significant between the blank plasmid group and the H/R group (p>0.05).Conclusion(1) Hypoxia/reoxygenation can induce the injury of oxidative stress and apoptosis of cardiomyocytes.(2) IMD gene has a particular position and function by transfecting into cells.(3) The protective effect of IMD against injury of cardiomyocytes by decreasing the oxidative stress and inhibiting the apoptosis of cardiomyocytes.