| Objective: Myocardial ischemia-reperfusion injury is an important cause of death from acute myocardial infarction.Oxidative stress injury is one of the important pathogenesis of reperfusion injury.Oxidative stress injury can cause myocardial cell death and reduce the benefits of reperfusion therapy.Therefore,it is of great significance to prevent reperfusion injury,explore the protective effect of traditional Chinese medicine on reperfusion injury,and provide effective theoretical and experimental basis for the prevention and treatment of ischemic cardiomyopathy.Previous studies have suggested that Wutou Chishizhi Pill can inhibit apoptosis and reduce reperfusion injury in rats with acute myocardial infarction,but the specific mechanism has not been fully elucidated.Autophagy is one of the cell's self-protection mechanisms.Existing evidence shows that autophagy plays a “double-edged sword” role in myocardial ischemia-reperfusion injury.Excessive autophagy triggers the occurrence of apoptosis and necrosis of myocardial cells.Moderate autophagy can play a role in prolonging the viability of myocardial cells and protecting myocardial function.The regulation of autophagy involves multiple signaling pathways,including the Erk1/2/Nrf2 signaling pathway.Erk/Nrf2 signaling pathway can induce the up-regulation of endogenous cytoprotective genes,including antioxidant and anti-inflammatory related proteins.Existing research suggests that under the condition of oxidative stress,the Erk1/2/Nrf2 signaling pathway can play a role in synergistic elimination of ROS by antioxidant substances in the body by activating autophagy.Wutou Chishizhi Pill has protective effects on myocardial cells in rats with acute myocardial infarction,but its mechanism of action remains to be further studied.Autophagy,mitochondrial damage,and oxidative stress are all important mechanisms of reperfusion injury,and the three are closely related.Therefore,the safety of autophagy can be judged from related indicators of mitochondrial damage and oxidative stress.On this basis,on the premise of confirming the protective effect of autophagic myocardial cell using multi-parameters,in terms of cell viability,apoptotic morphology,and expression of apoptosis-related proteins,the in vitro level of Wutou Chishizhi Pill under oxidative stress conditions by enhancing autophagy and reducing myocardial cell oxidative stress injury,the medicine was explored for its effect on Erk1/2/Nrf2/HO-1 pro-survival signaling pathway,its relationship with autophagy,and its potential role for clinical application“Wenyang” methods of ischemic heart disease provides experimental evidence.Research content: This experiment uses the H9C2 myocardial cell line as the experimental object.(1)Use H2C2 to stimulate H9C2 to construct oxidative stress model.(2)CCK-8 method was used to determine the viability of H9C2 cells,Levels of lactate dehydrogenase(LDH),Superoxide dismutase,SOD,and cardiac malondialdehyde(MDA)were also measured.immunofluorescence method was used to observe the morphology of nuclear chromatin,Western blot method was used to detect apoptosis-related proteins bax and bcl-2,and the autophagy inhibitor Baf A1 was used for intervention.To observe the protective effect of Wutou Chishizhi Pill decoction medicated serum on H9C2 cells.(3)Erk1/2 inhibitor was used to investigate the regulation of autophagy by Wutou Chishizhi Pill through Erk1/2/Nrf2/HO-1 signal transduction pathway.Results:(1)Establishment of oxidative stress model and the protective effect of Wutou Chishizhi Pill on H9C2 myocardial cell induced by hydrogen peroxide:The results of CCK-8 showed that the EC50 of hydrogen peroxide was?400 ?mol/ L.Therefore,H9C2 cells were induced by 400 ?mol / L hydrogen peroxide for 4 hours to construct a cell model of oxidative stress injury.Compared with the control group,the cell viability of the model group decreased significantly(P <0.01),LDH and MAD levels increased significantly(P < 0.01),SOD level decreased significantly(P < 0.01);the cell viability of the positive control group and the experimental group was significantly higher than that of the model group(P <0.05,P <0.01);the cell viability of the experimental group was higher than the positive group(P <0.05),this effect can be eliminated by the autophagy inhibitor Baf A1.The results of immunofluorescence showed that hydrogen peroxide could promote the apoptosis of H9C2 cells.Both Wutou Chishizhi Pill and Shenfu decoction could inhibit apoptosis,and the nucleus was completely regular;after adding the autophagy inhibitor Baf A1,apoptosis in the model group increased,nucleus ruptured,and apoptotic cells increased.Western blot results showed that compared with the control group,the expression of HO-1 and Bcl-2 in the model group decreased significantly(P <0.01);the expression of Bax increased significantly(P <0.01).Compared with the model group,theexpression of HO-1 and Bcl-2 in the experimental group and the positive control group increased significantly(P <0.01);the expression of Bax decreased significantly(P <0.01).Compared with the positive control group,Bax and Bcl-2 protein expressions in the experimental group were not significantly different,and HO-1 protein expression was significantly increased(P <0.01).The autophagy inhibitor Baf A1 could eliminate this effect.Compared with the control group,the expression level of LC3-? in the model group decreased significantly(P <0.01).Compared with the model group,the expression level of LC3-? in the experimental group and the positive control group increased significantly(P <0.01).The results showed that autophagy was inhibited in the model group and the activation of autophagy was promoted in the experimental group and the positive group.(2)Wutou Chishizhi pill activates Erk1/2/Nrf2/HO-1 signaling pathway,activates autophagy,and protects H9C2 myocardial cells damaged by hydrogen peroxide:CCK-8 results showed that compared with the control group,the cell viability of the model group decreased significantly(P <0.01);The cell viability of the positive control group and the experimental group was significantly higher than that of the model group(P<0.05,P<0.01);the cell viability of the experimental group was higher than that of the positive control group(P <0.05),and this result could be eliminated by GDC-0994.Western blot results showed that compared with the control group,the phosphorylation level of Erk1/2 in the model group decreased significantly(P <0.01);the expression levels of Nrf2,HO-1,Bcl-2,and LC3-? decreased significantly(P <0.01),and the expression levels of Bax increased significantly(P <0.01).Compared with the model group,the phosphorylation level of Erk1/2 in the experimental group and the positive control group was significantly increased(P <0.001);the expression levels of Nrf2,HO-1,Bcl-2,and LC3-? were significantly increased(P <0.01),Bax expression decreased significantly.Compared with the positive control group,the phosphorylation level of Erk1/2 in the experimental group was significantly increased(P <0.01),and the expression of Nrf2 and HO-1 proteins was significantly increased(P <0.01).There was no difference in Erk1/2protein expression between the groups.Erk1/2 inhibitor GDC-0994 can inhibit the Erk1/2phosphorylation induced by Wutou Chishizhi Pill decoction and Shenfu decoction(P<0.01),eliminate the up-regulation effect of Wutou Chishizhi Pill decoction on Nrf-2,HO-1,Bcl-2,LC3-? and down-regulation effect on Bax.Conclusion:(1)The oxidative stress-autophagy pathway is involved in myocardial ischemia-reperfusion injury,resulting in a time-dependent decrease in the vitality of myocardial cell,and the consumption of antioxidant enzymes in myocardial cell,the up-regulation of apoptosis-related proteins,leading to myocardial cell apoptosis.The medicated serum of Wutou Chishizhi Pill decoction can protect myocardial cell damage caused by hydrogen peroxide,inhibit Bax expression,promote Bcl-2 and LC-3 protein expression,promote SOD generation and reduce LDH and MAD levels,and promote cell autophagy.This process can be eliminated by the autophagy inhibitor Baf A1,it is shown that autophagy is a protective mechanism for protecting myocardial cell from oxidative stress injury.The medicated serum of Wutou Chishizhi Pill can promote the expression of Nrf-2and HO-1 proteins and promote the expression of LC3 and Bcl-2 proteins by activating the Erk1/2 signaling pathway.In the presence of the inhibitor GDC-0994,Nrf2,HO-1,LC3,SOD level.Bcl-2 expression was down-regulated,and Bax,MDA and LDH levels increase expression was up-regulated.It is suggested that it is possible to promote the autophagy of myocardial cell and exert anti-oxidative stress by up-regulating the Erk1/2/Nrf2/HO-1signaling pathway.However,there are multiple targets and multiple pathways in the intervention of Wutou Chishizhi Pill on myocardial cell autophagy,and the specific mechanism remains to be further studied. |
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