运动预适应提高心肌缺血耐受性的自噬调控机制研究

The ischemic myocardial injury is a common clinical disease; sometimes relative myocardial ischemia injury occurs in sports training. Exercise preconditioning can alleviate injury of ischemic myocardial, but the mechanism of myocardial protection induced by exercise preconditioning has not been completely clarified yet. Dates show that exercise may regulate autophagy to mediate its myocardial protection effects, but law and mechanism of exercise regulate myocardial autophagy, and cardioprotection of exercise preconditioning mediated by autophagy are not very clear. For the first time the subject will systematically study the influence and the mechanism of different intensity of exercise training on rats’ myocardial autophagy, and then study the autophagy mediated effects and molecular mechanism of exercise preconditioning. First, this work will establish SD rat model of different exercise intensity training; will detect myocardial autophagosome by electron microscopic; will detect expression of myocardial LC3, Beclin1, Atg5 etc.by RT-PCR and Western blot; will detect myocardial energy metabolism, changes of oxidative stress and endogenous factor; will investigate the mechanism of exercise training regulation autophagy; further will use inhibitors to ascertain the effects and disciplines of autophagy in exercise preconditioning; inbitors and protein kinase activity detection technology will be used to explore the mechanism and the signal transduction pathway of autophagy in the ischemia myocardium induced by exercise and ischemia reperfusion myocardium after exercise preconditioning; will explore the molecular mechanism of autophagy mediated exercise preconditioning.

缺血性心肌损伤是临床常见疾病，运动中时有心肌相对缺血损伤发生，通过运动预适应可以减轻缺血心肌的损伤，运动预适应心肌保护作用机制尚未完全阐明。资料表明：运动可能调控自噬来介导其心肌保护作用，但运动调控心肌自噬的规律与机制，及自噬介导运动预适应心肌保护作用尚不清楚。本课题首次系统研究不同强度的长期运动训练对大鼠心肌自噬的影响和机制，进而研究自噬在运动预适应中的介导作用和分子机制。首先拟建立不同运动强度的SD大鼠模型，电镜检测心肌自噬泡，RT-PCR和Western blot检测心肌LC3、Beclin1、Atg5等的表达，检测心肌中能量代谢、氧化应激及内源性因子的变化，探讨运动调控自噬的机制，进而应用自噬抑制剂探明自噬在运动预适应中的作用和规律，利用蛋白激酶技术和蛋白激酶活性检测技术研究预适应后运动性缺血心肌及缺血再灌注心肌自噬的机制及信号转导途径，深入探索自噬介导运动预适应的分子机制。

本研究主要解决运动预适应对心肌自噬的调控作用与机制，及其在抗心肌缺血再灌注损伤中的作用与机制。首先系统研究不同强度的长期运动训练对大鼠心肌自噬的影响和机制，进而研究运动预适应通过自噬调控产生心肌保护作用与机制。发现：①运动强度和运动时间是诱发心肌自噬的因素，低中强度运动没有引起明显的大鼠心肌自噬发生，高强度运动诱导的自噬随着时间进程而发展，心肌自噬的发生具有强度依赖性和时间依赖性；②运动训练过程中大鼠心肌能量代谢与自噬互相调节，低强度和中等强度的运动训练使能量代谢与自噬都保持在一个适度提高的水平，共同维持心脏功能在一个良好的水平；高强度运动训练启动能量调节通路上调自噬，且高强度运动训练诱导自噬水平的提高并不能补偿能量代谢的失衡，导致心脏功能受损；③氧化应激与自噬之间有一个相互调控，但因运动强度而异。中低强度运动训练氧化应激在心肌细胞自噬的调控过程中并未起到主导作用。高强度运动心肌氧化应激损伤促进心肌细胞自噬的产生，同时自噬的增多促进了抗氧化酶的消耗，形成恶性循环；④运动预适应可以引起缺血心肌细胞自噬水平的适度上调，以此改善心肌细胞代谢和收缩功能，从而对运动性心肌缺血和心肌缺血再灌注损伤中的心脏起保护作用。EP通过刺激ATF6释放、增加内源性NOS活性，适当提升NO水平，减轻缺血心肌中钙超载，减轻氧化应激损伤，从而调节自噬至一个适度的水平。⑤运动预适应启动了PI3K/Akt/mTOR信号通路、NF-κB信号通路、p53/自噬信号通路和JAK/STAT信号通路调控自噬，从而产生心肌保护作用。