The Effects Of Adiponectin Against Oxidative Stress Induced Endoplasmic Reticulum Stress Injury In Cultured Rat Neonatal Cardiomyocytes

Objective To investigate the effects of adiponectin on endoplasmic reticulum stress injury of the 3-4 days SD rat cardiomyocytes, which was induced by the intervention of H2O2.Methods Primary cardiomyocytes were 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 7 groups:①:control;②H2O2 (100μmol/L,2h);③:APN(30μg/ml)+H2O2;④:APN+AraA(0.5mmol/L,1h)+ H2O2;⑤:H2O2+AraA; The change of morphology of cardiomyocytes was observed by electron microscope. The content of LDH was detected by chemistry chromatometry; the cardiomycocytes apotosis was detected by agarose gelelectrophoresislevels and Annexin V-FITC/PI flow cytometry. The expression of GRP78, Caspase-12, CHOP, JNK, AMPK was detected Western blot and RT-PCR.Results(1) The indentification of cardiomyocytes:most cells displayed green fluorescence under fluorescent microscopy, which accords with is the characteristic 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 APN+H2O2 APN+AraA+H2O2 the cells have minor damage,the pulse is stronger than groupⅡ.In APN+ AraA+H2O2 group the morphological changes is between APN+H2O2 group and H2O2 group,and there is no significant changes between H2O2 group and APN+AraA+H2O2 group.(3) Comparison of the levels of LDH in different groups:When treated by H2O2 groupⅠ(353.33±50.32);groupⅡ:(1344.66±138.51), which was increased significantly compared with groupⅠ, p<0.05; groupⅢ(686.67±61.1), which was decreased significantly compared with groupⅡ, p<0.05;groupⅣ(914.33±103.12), 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 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, groupⅢand groupⅤ.(5) 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 69±5.29% and 70.67±6.43% 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 43.63±3.85%, there is statistically significant compared with groupⅡ, p<0.05; in groupⅣ, it's apoptosis rate is 59.87±4.44%, higher than groupⅢand lower than groupⅡ, p<0.05.(6) Expression of GRP78 and Caspase-12mRNA:RT-PCR results show the expression of GRP78 and caspase-12 mRNA had increased significantly in the H2O2 group. In the H2O2+APN group, the indexes above improved obviously compared with the H2O2 group (p<0.05). However, H2O2+APN+AraA group could aggravated the protective effects against oxidative stress injury (p<0.05)(7) Expression of GRP78, Caspase-12, CHOP, JNK protein:Western blot results show the expression of GRP78,CHOP, JNK protein had increased significantly in the H2O2 group. In the H2O2+APN group, the indexes above improved obviously compared with the H2O2 group (p< 0.05). However, H2O2+APN+AraA group aggravated the protective effects against oxidative stress injury (p<0.05)Conclusion(1)Oxidative stress can induce endoplasmic reticulum stress in rat cardiomyocytes.(2)Adiponectin can attenuate endoplasmic reticulum stress and play a protective role in the apoptosis of cadiomyocytes.(3)Adiponectin can protect cardiomyocytes from the apoptosis induced by oxidative stress through AMPK pathway.