Study on the Mechanism of Electroacupuncture Preconditioning Alleviating Myocardial Ischemia-Reperfusion Injury via MG53 Inhibition of the RIPK1/RIPK3/MLKL Necroptotic Pathway

TANG Yue; CHEN Ankun; LIU Xin; LOU Yating; YU Jie; ZHANG Chengcheng

Abstract：Objective To investigate the effects and underlying mechanisms of electroacupuncture preconditioning on myocardial ischemia-reperfusion injury(MIRI).Methods C57BL/6J mice were randomly divided into a sham group,model group,and electroacupuncture group,with 5 mice in each group.Mitsugumin 53(MG53)gene knockout(MG53-/-)mice were randomly divided into an MG53-/-model group and an MG53-/-electroacupuncture group,with 5 mice in each group.The electroacupuncture group and MG53-/-electroacupuncture group received electroacupuncture preconditioning at the"Neiguan"acupoint(PC6)for 7 consecutive days before model establishment,while the other groups were anesthetized and kept in a supine position as controls.On day 8,the MIRI model was established by ligating the left anterior descending coronary artery of the mice for 30 minutes,followed by 24 hours of reperfusion,after which tissue samples were collected.Western blot was performed to detect proteins including MG53,RIPK1,RIPK3,p-RIPK and MLKL.RT-qPCR was performed to detect mRNA expression of RIPK1,RIPK3,and MLKL.ELISA was used to measure myocardial tissue levels of ATP and serum levels of LDH,CK-MB,cTnT,MDA,SOD,caspase-1,IL-1β,and IL-18.TUNEL staining was performed to determine the myocardial cell apoptosis rate.Echocardiography was used to assess left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS).Myocardial infarction area was assessed using TTC staining.Mitochondrial ultrastructure was observed under an electron microscope,and histopathological changes in myocardial tissue were assessed using HE staining.Results In this study,electroacupuncture preconditioning alleviated MIRI effectively through multiple mechanisms.Compared with the model group,electroacupuncture preconditioning significantly improved cardiac function,evidenced by increased LVEF and LVFS levels(P<0.01),and enhanced ATP levels in myocardial mitochondria(P<0.05).It reduced serum levels of CK-MB,LDH,and cTnT(P<0.01),decreased the myocardial apoptosis rate(P<0.01),and caspase-1 expression(P<0.01),minimized myocardial infarction area(P<0.01),alleviated histopathological damage to myocardial tissue,protected mitochondrial structure,and inhibited inflammation and oxidative stress(P<0.05).Moreover,electroacupuncture preconditioning promoted the expression of MG53 in myocardial tissue and serum(P<0.01)and suppressed the expression of p-RIPK protein,as well as RIPK1,RIPK3,and MLKL proteins and mRNAs(P<0.05).Compared with the model group,the MG53-/-model group showed a larger myocardial infarction area(P<0.01),elevated levels of myocardial injury markers CK-MB,LDH,and cTnT(P<0.01),and increased expression of p-RIPK、RIPK1,RIPK3,and MLKL at both the protein and mRNA levels(P<0.05).Conclusion Electroacupuncture preconditioning at the Neiguan acupoint effectively alleviates MIRI,protects myocardial tissue,and improves cardiac function.The underlying mechanism may involve the regulation of the RIPK1/RIPK3/MLKL necroptosis signaling pathway via MG53 protein.