Hydrogen Sulfide On The Oxygen Stress-induced Myocardial Injury And Its Mechanism Discussed

Objective:To investigate the effects of H₂S on injury of the 3-4 days SD rat cardiomyocytes,which was induced by the intervention of H₂O₂,and research the mechanism of H₂S when it played the role.Methods:Primary cardiomyocytes was obtained from neonatal rat and were cultured by enzymatic digestion methods.The morphology of neonatal rat cardiomyocytes was studied by phase contramicroscope.Its molecular markers were observed byα-actin immunocytochemistry. Primary 3～4 days cells were used in experiment,and they were randomly divided into 5 groups: groupⅠ:treated without intervention as the control;groupⅡ:H₂O₂ group:the final concentration of H₂O₂ was 200μmol/L,and it was cultured with cadiomyocytes for 2 hours; groupⅢ:NaHS +H₂O₂ group:the final concentration of NaHS was 1μmol/L,it was added in the medium and cultured with cardiomyocytes for 30 minutes,then was cultured with H₂O₂ for 2 hours;groupⅣ:NaHS+LY294002+H₂O₂ group:the final concentration of LY294002 was 10mmol/L,NaHS and it was added in the medium and cultured with cardiomyocytes for 30minutes,then was cultured with H₂O₂ for 2 hours groupⅤ:LY294002+H₂O₂ group:LY294002 was added in the medium and cultured with cardiomyocytes for 30 minutes,than cultured with H₂O₂ for 2 hours..The change of morphology of cardiomyocytes was observed by phase contramicroscope.The content of LDH was detected by chemistry chromatometry;the content of MDA was detected by TBA colouration method;the content of SOD was detected by xanthine oxidase method;the cardiomycocytes apotosis was detected by agarose gel electrophoresislevels and AnnexinⅤ-FITC/PI flow cytometry.Results:(1) The indentification of cardiomyocytes:most cells displayed green fluorescence under fluorescent microscopy,which accords with the characteristics of cardiomyocytes.(2) The morphology of cardiocyte in every group:In the H2O2 group,the cells grew poorly, pseudopods of cardiocytes are shorten and disappeared,the pulse is weakened.In groupⅢthe cells have minor damage,the pulse is stronger than groupⅡ.In groupⅣthe morphological changes is between groupⅠand groupⅡ,and there is no significant changes between groupⅡand groupⅣ.(3) Comparison of the levels of LDH in different groups:When treated by H₂O₂, groupⅠ(443±56);groupⅡ:(1890±152),which was increased,p<0.05;groupⅢ(884±51),which was decreased significantly compared with groupll,p<0.05;groupⅣ(1165±569),which was increased compared with groupⅢ,p<0.05,and was decreased compared with groupⅢ, p<0.05;for group V,there is no statistically significant compared with groupⅡ. (4) Comparison of the levels of MDA in different groups:When treated by H₂O₂, groupⅠ(9.42±1.52);groupⅡ:(28.05±1.86),which was increased,p<0.05;groupⅢ(12.33±1.38), which was decreased significantly compared with groupⅡ,p<0.05;groupⅣ(20.82±2.32),which was increased compared with groupⅢ,p<0.05,and was decreased compared with groupⅢ, p<0.05;for groupⅤ,there is no statistically significant compared with groupⅡ.(5) Comparison of the levels of SOD in different groups:When treated by H₂O₂, groupⅠ(22.28±2.51);groupⅡ:(10.49±1.10),which was decreased,p<0.05;groupⅢ(19.06±0.87), which was increased significantly compared with groupⅡ,p<0.05;groupⅣ(20.82±2.32),which was decreased compared with groupⅢ,p<0.05,and was decreased compared with groupⅢ, p<0.05;for group V,there is no statistically significant compared with groupⅡ,p> 0.05.(6) Camparison of the fluorescence intensity of calcium:when treated by H₂O₂,the fluorescence of groupⅡ(1542±392.51) is higher than groupⅠ(239±65.94),p<0.05;groupⅢ(785±226.75),which is dcreased significantly compared with groupⅡ,p<0.05;groupⅣ(1123±269.52),which was increased compared with groupⅢ,p<0.05,for groupⅤ,there is no statistically significant compared with groupⅡ.(7) Comparison of DNA ladder in different groups:DNA ladder was detected by agarose gel electrophoresislevels.DNA ladder can't be detected in groupⅠ;however,there is significant DNA ladder in groupⅡand groupⅤ;in groupⅢ,it can attenuate the form of DNA ladder markedly;in groupⅣ,the brightness of DNA ladder was between groupⅡ,groupllland groupⅤ.(8) The result of flow cytometry:The cells in groupⅠconcentrated in B3 district,and there is no distribution in B4 and B2 district;In groupⅡand group V,it shows lots of apoptosis cells, and there still was a few of necrosis cells,and the rate of apoptosis is 54.8±1.75 and 55.6±1.23 respectively,there is statistically significant compared with groupⅠ,p<0.05;in groupⅢ,it can reduce the distribution of cells in B2 district,that is to say,reduce the apoptosis and necrosis cells,and the rate of apoptosis decreased to 15.5±1.10,there is statistically significant compared with groupⅡ,p<0.05;in groupⅣ,it's apoptosis rate is 39.2±2.42,higher than groupⅢand lower than groupⅡ,p<0.05.Conclusion:(1) H₂O₂ can induce the injury and apoptosis of cardiomyocytes;(2) H₂S can release the injury and apoptosis and play a protective role on the apoptosis of cadiomyocytes;(3) LY294002,the inhibitor of PI3K/Akt channel,can inhibit the protective role of H₂S...