摘要
目的探究电针预处理对心肌缺血再灌注损伤(myocardialischemia-reperfusioninjury,MIRI)的影响与相关机制。方法C57BL/6J小鼠随机分为假手术组、模型组和电针组,每组5只;MG53基因敲除(MG53^(-/-))小鼠随机分为MG53^(-/-)模型组和MG53^(-/-)电针组,每组5只。电针组和MG53^(-/-)电针组在造模前连续7 d进行“内关”穴电针预处理,其余组麻醉后陪同仰卧固定。第8天建立MIRI模型,结扎小鼠左冠状动脉前降支30 min,再灌注24 h后取材。Westernblot检测肌细胞膜修复蛋白(MG)53、受体相互作用蛋白激酶1(RIPK1)、RIPK3、磷酸化(p)-RIPK、混合谱系激酶结构域样蛋白(MLKL)蛋白表达,RT-q PCR检测RIPK1、RIPK3、MLKLm RNA表达;ELISA检测心肌组织三磷酸腺苷(ATP)、血清乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白T(cTnT)、丙二醛(MDA)、超氧化物歧化酶(SOD)、半胱氨酸天冬氨酸蛋白酶(caspase)-1、白细胞介素(IL)-1β、IL-18水平;TUNEL法检测心肌细胞凋亡率;心脏超声评估左室射血分数(LVEF)和左室短轴缩短率(LVFS);TTC染色评估心肌梗死面积;电镜观察线粒体超微结构;HE染色观察心肌组织病理形态变化。结果在本研究中,电针预处理通过多种机制有效缓解了MIRI。与模型组比较,电针预处理显著改善了心脏功能,提高了LVEF和LVFS水平(P<0.01),增加了心肌组织线粒体ATP含量(P<0.05);降低了CK-MB、LDH和cTnT水平(P<0.01),降低了心肌细胞凋亡率(P<0.01)和caspase-1表达(P<0.01),缩小了心肌梗死面积(P<0.01),减轻了心肌组织病理损伤,保护了线粒体结构,并抑制了炎症因子和氧化应激(P<0.05)。此外,电针预处理能够促进心肌和血清中MG53蛋白的表达(P<0.01),并抑制p-RIPK蛋白与RIPK1、RIPK3、MLKL蛋白及m RNA的表达(P<0.05)。与模型组比较,MG53^(-/-)模型组的心肌梗死面积增大(P<0.01),心肌损伤标志物CK-MB、LDH、cTnT水平升高(P<0.01),p-RIPK蛋白表达增加,RIPK1、RIPK3、MLKL蛋白及mRNA的表达增加(P<0.05)。结论电针内关预处理可以有效缓解MIRI、保护心肌组织、改善心功能,其机制可能与电针通过MG53蛋白影响RIPK1/RIPK3/MLKL坏死性凋亡信号通路有关。
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.
作者
唐玥
陈安坤
刘欣
娄雅婷
郁洁
张程程
TANG Yue;CHEN Ankun;LIU Xin;LOU Yating;YU Jie;ZHANG Chengcheng(School of Acupuncture-moxibustion,Tuina and Rehabilitation,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国中医基础医学杂志》
2025年第7期1270-1278,共9页
JOURNAL OF BASIC CHINESE MEDICINE
基金
国家自然科学基金项目(82205295)
湖南省教育厅科学研究重点项目(23A0299)。
关键词
电针预处理
心肌缺血再灌注损伤
MG53
坏死性凋亡
线粒体
Electroacupuncture preconditioning
Myocardial ischemia-reperfusion injury
MG53
Necroptosis
Mitochondria
