EGCG调控自噬改善心肌缺血再灌注后无复流的机制研究

No-reflow after cardiac ischemia/reperfusion (I/R) severely demolishes the benefits of coronary revascularization in patients with acute myocardial infarction. Despite the recanalization of blocked epicardial coronary artery, dysfunction of myocardial tissue microcirculation may lead to myocardial no-reflow phenomenon with high mortality and unclear mechanism. Our previous data indicated that autophagy was promoted in animal myocardial no-reflow area accompanied by increased reactive oxygen species(ROS), neutrophil infiltration and the activation of NF-κB. Our preliminary study indicated that pretreatment with epigallocatechin gallate(EGCG)，the predominant catechin component of green tea, reduced no-reflow extent and inhibited autophagy. We therefore propose that EGCG decreases the expression of pro-inflammatory factors and neutrophils recruitment in no-reflow area after myocardial I/R as well as inhibits autophagy through ROS/NF-κB/Beclin 1 signaling pathway to reduce no-reflow. In this project, we use the method of animal no-reflow model to determine the relationship between EGCG and inflammation, ROS, autophagy as well as myocardial tissue injury. Meanwhile, we culture endothelial cells and NF-κB over-expressing endothelial cells in simulated I/R media to investigate the effect of different doses EGCG on the regulation of autophagy signaling pathway. By investigating the mechanism of EGCG on amelioration no-reflow by regulating autophagy, we sought to provide a theoretical foundation for therapeutic interventions of myocardial I/R-associated no-reflow.

心肌缺血再灌注（I/R）后无复流严重影响急性心肌梗死冠脉搭桥或介入治疗患者的预后。冠脉再通后心肌微循环障碍导致的无复流现象死亡率高，具体机制不明。我们前期研究显示动物心脏I/R后无复流区活性氧簇（ROS）增加，粒细胞浸润，NF-κB和自噬激活。预实验中绿茶多酚EGCG的应用可以减少无复流面积及抑制自噬。因此提出“EGCG通过减少I/R后无复流区促炎因子的分泌和白细胞募集，同时抑制ROS/NF-κB/Beclin 1信号转导通路抑制细胞自噬，最终减少心肌无复流”这一假说。本课题应用动物心肌I/R后无复流模型，观察EGCG与炎症反应、氧化应激、细胞自噬及无复流的关系。采用模拟I/R培养内皮细胞或稳定过表达NF-κB内皮细胞，观察不同剂量EGCG对细胞自噬信号转导通路的影响。从分子水平、组织水平和动物体内实验，探讨EGCG调控自噬改善心肌无复流的机制，为临床防治无复流提供理论依据和治疗靶点。

表没食子儿茶素没食子儿茶素没食子酸酯（EGCG）因其对心血管健康的益处而受到越来越多的关注。本研究探讨EGCG对心肌缺血/再灌注（I/RI）损伤保护作用的潜在分子途径。采用小鼠心肌I/RI模型和H2O2诱导心肌细胞损伤模型，观察EGCG的治疗作用。在体内心肌I/RI小鼠模型中，通过超声心动图参数射血分数（EF）和缩短分数（FS）水平、梗死面积、组织学评价和透射电镜（TEM）观察心肌组织损伤和自噬活性。体外模型采用MTT法、TUNEL法、流式细胞术、LC3免疫组化法检测细胞活力及表型变化。分别用qRT-PCR和western-blot检测Gm4419和DUSP5的表达。RNA结合蛋白免疫沉淀（RIP）、RNA下拉和染色质免疫沉淀（ChIP）分析证实了Gm4419对DUSP5的表观遗传调控。研究结果显示，EGCG能明显改善I/RI小鼠心肌功能，缩小梗死面积，提高细胞活力，抑制H2O2诱导的心肌细胞自噬活性。此外，在H2O2和心肌I/RI小鼠中观察到的高表达Gm4419被EGCG抑制，并且Gm4419的过度表达显著地取消了EGCG介导的心肌I/RI保护作用。在机制上，Gm4419通过招募EZH2表观遗传学抑制DUSP5，从而激活ERK1/2通路介导的自噬。此外，体内研究进一步证实，过度表达Gm4419介导的EGCG对心肌I/RI的保护作用被DUSP5敲除而增强，但被DUSP5过度表达削弱。总之，我们的研究结果表明EGCG通过调节lncRNA Gm4419/DUSP5/ERK1/2介导的自噬过程来保护心肌I/RI。