Effect Of Linggui Zhugan Decoction On Oxidative Stress Injury And Apoptosis Of Cardiac Myocytes Induced By Hydrogen Peroxide And Its Effect On Nrf2/ARE Pathway

Objective:To observe the effects of Linggui Zhugan Decoction on oxidative stress,apoptosis and expression of Nrf2/ARE signaling pathway-related proteins in myocardial cells induced by hydrogen peroxide（H₂O₂）and explore the anti-oxidative stress injury and its mechanism of Linggui Zhugan Decoction.Method:Myocardial cells were isolated and extracted by differential adherence method,pretreated with Linggui Zhugan decoction containing serum for 12 hours,and treated with hydrogen peroxide for 6 hours to induce oxidative stress injury model of myocardial cells.Myocardial cells were randomly divided into six groups:normal group,H₂O₂model group,blank rat serum（20%）group,Linggui Zhugan Decoction（5%,10%,20%）containing serum group.The myocardial cells were cultured with 20%normal rat serum,5%,10%and 20%for 12 hours.Except for the normal control group,the other groups were added with H₂O₂（100μmol/L）for 6hours to establish myocardial oxidative stress model.1.MDA content and LDH,SOD,GSH-Px activity in cardiomyocytes were measured to explore the effects of oxidative stress on cardiomyocytes.2.Influences on Inflammatory Factors in Cardiac Cells:TNF-α,IL-1βand IL-6 were detected by ELISA to explore the influences on inflammatory.3.The effect of apoptosis on cardiomyocyte apoptosis:morphological changes of apoptotic cells.The expression of bcl-2,bax,caspase-9 and caspase-8 protein in cardiomyocyte was detected by flow cytometry and ELISA.Western Blot assay was used to detect caspase-3,Cyt-c and myocardial cells in each group.4.Effect on ROS and membrane potential of myocardial cells:Flow cytometry was used to detect ROS content and fluorescence microscopy was used to observe the changes of mitochondrial membrane potential.;5.The expression of Nrf2/ARE signaling pathway related proteins,Nrf2,HO-1 and NQO-1 in cardiac myocytes.Result1.Effects of oxidative stress on cardiac myocytes:Compared with the normal control group,SOD and GSH-Px activities in the H₂O₂model group decreased significantly（P<0.01）,while MDA and LDH activities increased significantly（P<0.01）.Compared with the model group of H₂O₂,the serum containing Linggui Zhugan Decoction（10%,20%）significantly increased SOD,GSH-Px activity（P<0.01）,decreased MDA content and LDH activity（P<0.01）.2.Influences on myocardial inflammatory factors:ELISA results showed that the contents of TNF-α,IL-1βand IL-6 in myocardial cells of the model group were significantly higher than those of the normal control group（P<0.01）;comparing with the model group,the contents of TNF-α,IL-1βand IL-6 in myocardial cells of Lingguizhugan decoction containing serum group（5%,10%,20%）were significantly lower（P<0.01）.3.Effects on cardiomyocyte apoptosis:Compared with the normal control group,the apoptosis of cardiomyocyte was significantly increased（P<0.01）;the expression of Cyt-c,caspase-9,caspase-8,caspase-3 and Bax was decreased（P<0.01）;the expression of Bcl-2 was increased（P<0.01）;compared with the group of H₂O₂,the expression of Cyt-c,caspase-9,caspase-3,Caspase-8 and Bax was significantly decreased（P<0.01）;the expression of Bcl-2 was increased（P<0.01）.4.Effects on ROS and membrane potential of cardiac myocytes:Compared with the blank control group,the content of ROS in cells increased significantly,and the mitochondrial membrane potential was depolarized significantly（P<0.01）;Compared with the model group of H₂O₂,the serum group containing Lingguizhugan Decoction could significantly improve the depolarization of mitochondrial membrane potential and reduce the apoptotic rate（P<0.01）.5.The effect of Nrf2/ARE signaling pathway-related proteins on cardiac myocyte apoptosis:Compared with the normal control group,the expression of Nrf2,Ho-1,NQO-1 and Nrf2 protein increased（P<0.01）.Compared with the group of H₂O_2,the expression of Nrf2/ARE signaling pathway-related proteins increased.the expression of Nrf2,HO-1,NQO-1 and Nrf2 nuclear translocation in cardiac myocytes of the Lingguizhugan decoction containing serum group increased with the increase of dose.（P<0.01）.Conclusion:The serum containing Linggui Zhugan Decoction can increase the antioxidative stress response ability of cardiac myocytes,alleviate inflammation and reduce apoptosis of cardiac myocytes,and thus protect cardiac myocytes.The mechanism of action may be related to the regulation of proteins involved in the Nrf2/ARE signaling pathway.