【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。更多还原

【Abstract】 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 phoAMPK, AMPK, NLRP3, Cleaved caspase-1, IL-1β were 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-1β was 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-1 and 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.更多还原