内源性大麻素系统通过AMPK通路介导心肌线粒体自噬机制研究

Mitochondrial damage of cardiomyocytes is the main cause of the low survival rate in patients after resuscitation. We have preliminarily found that the activation of the endocannabinoid system (ECS) can raise the level of mitochondrial autophagy of myocardial cells, which can significantly reduce the myocardial injury after cardiac arrest. To explore the mechanism, our study aims to detect the monoacylglycerol cannabinoid (2-AG) level in plasma in patients after cardiopulmonary resuscitation, and do regression analysis with the prognosis and cardiac function of patients, in order to study whether ECS activation can improve cardiac function and reduce the risk of adverse cardiac events. Then, we make the cardiopulmonary resuscitation model by rat, by inhibition of endogenous cannabinoid hydrolase as well as determination of myocardial enzymes and mitochondrial autophagosome, to explore that whether the raise of 2-AG level can promote the myocardial mitochondrial autophagy, thereby reducing myocardial injury. Finally, we use the primary cardiomyocyte hypoxia model to inhibit receptors of endogenous cannabinoid system, and use the siRNA to down-regulate the AMPK, aiming to study whether ECS mediated myocardial mitochondrial autophagy through the AMPK pathway. We also use transcriptomics and proteomics to explore the regulation of AMPK downstream signal pathway. This study will provide new ideas and strategies for early clinical intervention of cardiac arrest.

心肌细胞线粒体损伤是造成心肺复苏后患者死亡的主要原因。我们前期研究发现，内源性大麻素系统（ECS）的激活可提高心肌细胞线粒体自噬作用，从而减轻心肺复苏后的心肌损伤。为探讨其机制，本项目拟通过测定心肺复苏后患者血浆单酰基甘油类大麻素（2-AG）水平，并与患者预后和心脏功能做回归分析，研究ECS激活能否提高心脏功能，减少心脏不良事件风险；利用大鼠心肺复苏模型，通过抑制内源性大麻素水解酶并测定心肌酶学和线粒体自噬小体，研究提高2-AG水平是否能促进心肌线粒体自噬作用，从而减轻心肌损伤；最后，利用原代心肌细胞缺氧模型，通过抑制内源性大麻素系统受体，并使用siRNA下调AMPK，研究ECS是否通过AMPK通路介导心肌线粒体自噬作用，并采用转录组学和蛋白质组学，探讨AMPK下游信号通路调控。本课题将会为心肺复苏患者早期干预治疗和改善预后提供新的思路和理论依据。

心脏骤停-心肺复苏（CA/CPR）后心功能障碍是引起复苏后患者存活率低的最主要原因之一。研究发现，内源性大麻素系统（ECS）水解酶单酰基甘油酯酶（MAGL）通过水解2-酰基甘油（2-AG）对心脏可能产生保护作用。本研究结合大鼠心脏骤停-心肺复苏模型，在自主循环恢复后1分钟给予MAGL抑制剂URB602，观察大鼠复苏后24、48、72和168小时大鼠生存率，评估心脏功能、神经功能及内环境情况。同时，检测复苏后6小时血浆丙二醛（MDA）含量、过氧化物歧化酶（SOD）活性评估氧化应激反应；检测复苏后6小时血浆髓过氧化物酶（MPO）含量、复苏后2小时肿瘤坏死因子（TNFα）含量、心肌丙二醛（MDA）含量、花生四烯酸及其代谢产物含量，并结合血浆肌酸激酶同工酶（CKMB）活性和心肌病理变化，综合评估CA/CPR心脏氧化应激损伤。进一步，通过心肌MDA含量和EF相关性分析，证明心肌氧化应激与心功能损伤密切相关。最后，观察复苏后6小时心肌线粒体超微结构判断线粒体结构损伤；检测复苏后15分钟心肌线粒体吸光度、线粒体肿胀率变化、线粒体基础耗氧量等判断线粒体功能和能量代谢，验证CA/CPR心肌线粒体损伤与复苏后心功能不全的关系。本研究最终证明：MAGL抑制剂URB602一方面通过激活心肌细胞CB1，调控内源性大麻素和花生四烯酸代谢而发挥心肌保护作用，提高复苏后生存率；另一方面可降低线粒体结构和功能损伤，减少线粒体基础耗氧量及心肌脂质过氧化，通过保护线粒体结构和功能，达到保护CA/CPR心脏功能的作用。