二甲双胍激活腺苷酸活化蛋白激酶抑制心肌缺氧再灌注损伤介导的NOD样受体蛋白3炎症体活化的研究  

Metformin Postconditioning Attenuates Hypoxia/Reperfusion Injury in Isolated Rat Heart Through AMPK-mediated Inactivation of NLRP3 Inflammasomes

作　　者：杨柳[1] 余鹏[1] 邹芳[1] 蔡霞[1] 黄艳婷 施星 时曼莹 张勤 江艳娟 赖晓阳[1] YANG Liu;YU Peng;ZOU Fang;CAI Xia;HUANG Yanting;SHI Xing;SHI Manying;ZHANG Qin;JIANG Yanjuan;LAI Xiaoyang(Department of Endocrinology and Metabolism,The Second Affiliated Hospital of NanChang University,Nanchang 330006,JiangXi,China)

机构地区：[1]南昌大学第二附属医院,内分泌代谢科,江西省南昌市330006

出　　处：《中国循环杂志》2020年第9期927-933,共7页Chinese Circulation Journal

基　　金：国家自然基金项目(81760050);江西省科技厅青年科学基金(20192ACBL21037)。

摘　　要：目的:评价腺苷酸活化蛋白激酶(AMPK)介导的NOD样受体蛋白3(NLRP3)炎症体活化在二甲双胍后处理保护心肌缺氧再灌注(H/R)损伤中的作用及机制。方法:选取健康雄性SD大鼠64只,进行麻醉,开胸直接取心脏,将心脏悬挂在Langendoff灌注装置上建立离体心肌缺氧再灌注模型。随机分为4组(n=16):持续灌注组、缺氧再灌注组(H/R组)、二甲双胍后处理组(MET组)、二甲双胍后处理+AMPK抑制剂(CC)组(MET+CC组)。各组进行相应处理后,监测血流动力学指标,测定心肌梗死面积,观察心肌细胞超微结构,ELIS法测定心肌组织内乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)水平,心肌组织原位缺口末端标记法(TUNEL)染色检测细胞凋亡发生;蛋白免疫印迹(Western blot)法测定磷酸化AMPK、AMPK、NLRP3、活化含半胱氨酸的天冬氨酸蛋白水解酶-1(caspase-1)、IL-1β和GAPDH蛋白表达水平。结果:与持续灌注组比较,H/R组心肌梗死面积增大(P<0.05),LDH、CK-MB水平增加(P<0.05),心肌细胞凋亡率显著升高(P<0.05);与H/R组比较,MET组心肌梗死面积缩小(P<0.05),LDH、CK-MB水平降低(P<0.05),心肌细胞凋亡率下降(P<0.05),磷酸化AMPK/AMPK比值显著增加(P<0.05),而NLRP3、活化caspase-1和IL-1β表达显著下降(P<0.05);与MET组比较,MET+CC组抑制AMPK激活后心肌梗死面积进一步增大(P<0.05),LDH和CK-MB水平进一步升高(P<0.05),心肌细胞凋亡率明显升高(P<0.05);磷酸化AMPK/AMPK比值降低(P<0.05),而NLRP3、活化caspase-1和IL-1β表达显著升高(P<0.05)。结论二甲双胍通过激活AMPK信号通路,抑制心肌内NLRP3炎症体活化,从而减轻离体大鼠心肌缺氧再灌注损伤。Objectives:To study the role of metformin postconditioning in myocardial hypoxia reperfusion(H/R)injury and mechanism related to Amp-activated protein kinase(AMPK)mediated NOD-like receptor protein3(NLRP3)inflammasomes inhibition in the isolated rat H/R injury model.Methods:After anesthesia,the rat hearts were excised,and suspended on the Langendorff perfusion device to establish the isolated myocardial hypoxia-reperfusion model.Sixty-four male rats were randomly divided into 4 groups(n=16):continuous perfusion group,hypoxia/reperfusion(H/R)group,metformin postconditioning(MET)group,metformin postconditioning+AMPK inhibitor(MET+CC)group.After corresponding treatment,hemodynamics were monitored,myocardial infarct size and ultrastructure of myocardium were observed.The levels of lactate dehydrogenase(LDH)and creatine kinase(CK-MB)were measured,myocardial apoptotic index was measured by TUNEL.Protein expressions of pho-AMPK,AMPK,NLRP3,Cleaved caspase-1,IL-1βwere determined by Western blot.Results:Compared with the Continuous perfusion group,myocardial infarct size,LDH and CK-MB levels and index of apoptosis were significantly increased in the H/R group(all P<0.05).Compared with the H/R group,myocardial infarct size,the content of LDH and CK-MB levels,myocardial apoptotic index were significantly decreased,while the protein expression of pho-AMPK/AMPK was significantly up-regulated,and the expression of NLRP3,Cleaved caspase-1 and IL-1βwas significantly down-regulated in the MET group(all P<0.05).Compared with the MET group,the myocardial infarction area,LDH and CK-MB levels were increased(all P<0.05),the expressions of pho-AMPK/AMPK were down-regulated(P<0.05),and the expression of NLRP3,Cleaved caspase-1and IL-1β was significantly up-regulated in the MET+CC group(all P<0.05).Conclusions:Metformin could attenuate H/R injury in isolated Langendorff rat hearts and its mechanism was related to AMPK mediated inactivation of NLRP3 inflammasomes.

关 键 词：二甲双胍 心肌缺氧再灌注损伤 腺苷酸活化蛋白激酶 NOD样受体蛋白3 炎症小体 

分 类 号：R54[医药卫生—心血管疾病]