益气活血法调控硫化氢-自噬通路抗心肌缺血再灌注损伤的机制研究

Myocardial ischemia reperfusion （IR）injury treats human’s life gravely,while the concrete mechanism has still not been clarified.H2S is concerned with protecting myocardial ischemia injury by means of regulating cell’s autophagy. PI3K/Akt/mTOR signaling pathway which could be influenced by H2S plays a significant role in mediating autophagy.Our previous research indicated that the Jiawei Danshen decoction (JDT) based on Yiqi Huoxue therapy offers protection in the IR injury preprocessing of myocardial cells.Updated study also revealed that JDT increase cystathionine-γ-lyase （CSE）expression of myocardium and accelerate production of H2S as well as reduce key gene expression of autophagy.So we infer that JDT protects myocardium due to stimulate PI3K/Akt/mTOR by promoting auto H2S synthesis and regulate moderate autophagy post-IR.We discuss issues as follows in this research:①whether H2S affects myocardial post IR injury by mediating PI3K/Akt/mTOR pathway;②whether JDT regulates autophagy by promoting CSE/H2S metabolism so as to activate PI3K/Akt/mTOR in order to protect myocardial after IR injury.This study aims at enriching connotations of pathogenesis of Coronary disease with “qi deficiency and blood stasis” syndrome and corresponding therapeutic principle of “Yiqi Huoxue therapy”,seeking new curative targets and drugs for myocardial IR injury.

心肌缺血再灌注（IR）损伤严重影响生命健康，其确切机制尚未明确。适度自噬是保护IR后心肌细胞的重要机制，硫化氢（H2S）可调节自噬，两者均与PI3K/Akt/mTOR信号通路关联。我们前期研究发现，以益气活血立法的加味丹参饮预处理具有模拟缺血预适应样心肌保护作用。近期初步实验表明：本方能上调IR心肌细胞H2S合成酶CSE表达，下调自噬基因表达。据此，我们推测“加味丹参饮通过促进内源性H2S合成，激活PI3K/Akt/mTOR，调控IR后细胞适度自噬保护心肌细胞”。本项目采用整体和体外细胞实验，探讨①H2S是否介导PI3K/Akt/mTOR 通路影响心肌IR后细胞自噬；②加味丹参饮是否通过促进CSE/H2S生成—激活PI3K/Akt/mTOR—调节自噬，发挥IR后心肌保护作用。本研究旨在丰富冠心病“气虚血瘀”病机和“益气活血”治法的科学内涵，为心肌IRI治疗寻找新的治疗靶点和治疗药物。

本项目旨在验证益气活血组方加味丹参饮（JWDS）通过促进内源性H2S合成，激活PI3K/Akt/mTOR，调控心肌缺血再灌注损伤（MIRI）细胞适度自噬，发挥心肌保护作用。围绕目标，研究内容包括2部分：（1）以SD大鼠MIRI为模型，探究IRI后H2S代谢与自噬相关变化，证实JWDS的保护机制与调节H2S/CSE信号通路介导的自噬有关。（2）通过细胞实验复制H9C2心肌细胞缺氧复氧模型，分别以H2S合成酶CSE和PI3K/Akt/mTOR通路为切入点，阐明JWDS介导H2S生成，激活PI3K/Akt/mTOR通路，调节心肌细胞适度自噬，保护MIRI的作用机制。结果显示：（1）在体实验：与IRI模型组大鼠相比，JWDS组和NaHS组IRI模型大鼠心肌细胞损伤减轻，PPG组细胞排列紊乱，破坏严重；JWDS可明显上调心肌组织CSE表达，血清H2S生成增多，LC3A/B、Beclin1、ATG5表达降低；反之与PPG合用的JWDS组，CSE表达明显降低，血清H2S减少，并与JWDS组相比，LC3A/B、Beclin1表达增高，差异有统计学意义。表明JWDS对IRI大鼠心肌损伤具有保护作用，其机制与促进内源性H2S生成介导细胞自噬活性从而保护心肌细胞结构有关。（2）细胞实验：H9C2 IRI细胞组细胞数量少，形态不规则；电镜下自噬小体增多，线粒体破坏； NaHS与JWDS组细胞损伤减轻，自噬小体减少；与模型组比较，3-MA自噬抑制剂组CSE表达明显增加，RAPA组表达趋势与其相反，IR细胞自噬情况CSE的表达呈负相关；进一步检测PI3K-Akt-mTOR信号通路的变化，IRI组表达降低，而Beclin-1和LC3Ⅱ的蛋白表达明显上升，表明自噬水平提高，与模型组相比JWDS组和3MA组PI3K-3活化明显，而PPG组表达明显减少，自噬相关蛋白表达提高；证实PI3K-Akt-mTOR信号通路与自噬表达呈负相关，H2S可能介导该通路调控自噬（详见结果整理与分析）。因此本项目进一步证实了JWDS保护MIRI的多靶点效应，明确了冠心病“气虚血瘀”病机和“益气活血”治法的科学内涵。项目组将进一步从临床和实验证实益气活血法抗IRI作用，探索中医药防治MIRI病理生理机制，进一步研究JWDS配伍优化及确切保护机制，为复方中药的临床运用提供新思路和新方法，具有良好的社会效益和临床应用前景。