Adiponectin Oxygen Stress Due To Its Mechanism Of Myocardial Injury

Objective: To investigate the effects of adiponectin on injury of the 3-4 days SD rat cardiomyocytes, which was induced by the intervention of H₂O₂, and research the mechanism of adiponectin 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 by a-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: adiponectin+H₂O₂ group: the final concentration of adiponectin was 30mg/L, it was added in the medium and cultured with cardiomyocytes for 18 hours, then was cultured with H₂O₂ for 2 hours; groupⅣ: adiponectin+AraA+H₂O₂ group: the final concentration of AraA was 1mmol/L, adiponectin and it was added in the medium and cultured with cardiomyocytes for 18 hours, then was cultured with H₂O₂ for 2 hours; groupⅤ: AraA+H₂O₂ group: The adiponectin and AraA was added in the medium and cultured with cardiomyocytes for 18 hours, than cultured with H₂O₂ for 2 hours. The change of morphology of cardiomyocytes was observed by electron microscope. 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 Annexin V-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 and the electron microscope showed significant granular degeneration and necrosis, myofilament dissolved ,traveling disorder and a high degree of mitochondrial swelling and degeneration; however, most cells in groupⅢgrew well, and only a few of the cells appeared mild edema, and no significant bleeding lesions and granular degeneration,and there is no obvious swelling in mitochondrial(3) Comparison of the levels of LDH in different groups: When treated by H₂O₂ , groupⅠ(428±84);groupⅡ: (1712±218), which was increased, p<0.05; groupⅢ(830±111), which was decreased significantly compared with groupⅡ, p<0.05;groupⅣ(1546±162), 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.(4) Comparison of the levels of MDA in different groups: When treated by H₂O₂ , groupⅠ(9.79±3.52); groupⅡ: (32.69±15.16), which was increased, p<0.05; groupⅢ(12.75±3.93), which was decreased significantly compared with groupⅡ, p<0.05; groupⅣ(28.67±4.94),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(24.09±4.58); groupⅡ: (13.08±3.08), which was decreased, p<0.05; groupⅢ(19.99±3.12), which was increased significantly compared with groupⅡ, p<0.05; groupⅣ(14.87±2.34),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) 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 group V;in groupⅢ,it can attenuate the form of DNA ladder markedly; in groupⅣ, the brightness of DNA ladder was between groupⅡ, group V and groupⅢ.(7) The result of flow cytometry: The cells in groupⅠconcentrated in B3 district, and there is no distribution in B4 and B2 district; In groupⅡand groupⅤ, it shows lots of apoptosis cells, and there still was a few of necrosis cells, and the rate of apoptosis is 29.1±4. 78 and 27.5±3. 65 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 9.0±3. 30, there is statistically significant compared with groupⅡ, p<0. 05; in groupⅣ, it's apoptosis rate is 22.2±2. 99, higher than groupⅢand lower than groupⅡ, p<0. 05.Conclusion:(1) H₂O₂ can induce the injury and apoptosis of cardiomyocytes;(2) Adiponectin can reverse the injury and apoptosis and play a protective role on the apoptosis of cadiomyocytes;(3) AraA, the inhibitor of AMPK channel, can inhibit the protective role of adiponectin.