芪参活血颗粒激活MasR/PI3K-Akt-mTOR抑制自噬改善脓毒症心肌损伤机制研究

The mortality of myocardial injury induced by sepsis is high. Blood stasis syndrome is the key pathogenesis of myocardial injury in sepsis. We have found that Qishenhuoxue granule could reduce the expression of classical ACE-AngⅡ-AT1R axis of renin angiotensin system(RAS) , enhance the expression of new ACE2-Ang(1-7)-MasR axis and inhibit autophagy in heart. But the molecular mechanism after MasR of Qishenhuoxue granule which plays the role of promoting blood circulation for removing blood stasis is not clear. Recent studies discovered that the PI3K-Akt-mTOR pathway which inactivates autophagy is the downstream pathway of the ACE2 axis. By the methods of molecular biology, we will study that: (1) The activation of Qishenhuoxue granule to the MasR pathway by observing the effects of this recipe on the expression of MasR, PI3K, Akt, p-Akt, mTOR and p-mTOR in HL-1 cells or myocardial tissue of rats induced by lipopolysaccharide (LPS); (2) The regulatory sites of this recipe by observing the effects of drugs on myocardial autophagy and myocardial injury markers through inhibiting MasR, PI3K and mTOR in HL-1 cells and rats induced by lipopolysaccharide respectively. We elucidate the mechanism by which this recipe activates MasR/PI3K-Akt-mTOR pathway to inhibit myocardial autophagy and provide new targets for the treatment of myocardial injury in sepsis by promoting blood circulation for removing blood stasis.

脓毒症合并心肌损伤病死率极高，血瘀证是其关键病机。我们研究表明芪参活血颗粒通过下调促进“血瘀”形成的RAS经典轴ACE-AngⅡ-AT1R、上调改善“血瘀”的RAS新轴ACE2-Ang(1-7)-MasR及抑制心肌自噬改善脓毒症心肌损伤。但该方“活血化瘀”的MasR后分子机制尚不清楚。新近发现PI3K-Akt-mTOR为MasR下游通路，其激活抑制自噬。本课题应用分子生物学等方法研究：(1) 芪参活血颗粒对MasR后通路活化作用：该方对LPS处理的心肌细胞和大鼠MasR、PI3K、Akt、p-Akt、mTOR、p-mTOR表达影响；(2)该方的调控位点：分别抑制LPS处理的心肌细胞和大鼠心肌MasR、PI3K、mTOR，观察该方对心肌自噬和心肌损伤的影响，阐明该方激活MasR/PI3K-Akt-mTOR通路抑制心肌自噬改善脓毒症心肌损伤的机制。为活血化瘀疗法治疗脓毒症心肌损伤提供新靶点。

脓毒症合并心肌损伤病死率高，血瘀证是其关键病机。我们既往的研究表明芪参活血颗粒通过下调促进“血瘀”形成的RAS经典轴ACE-AngⅡ-AT1R、上调改善“血瘀”的RAS新轴ACE2-Ang(1-7)-MasR、抑制心肌细胞自噬改善心肌损伤。新近研究表明PI3K-Akt-mTOR是MasR后通路，激活后可改善自噬。本课题通过细胞和体内实验阐述该药对MasR/PI3K-Akt-mTOR通路及自噬的影响，并探寻其作用靶点。本课题利用LPS处理H9C2心肌细胞复制脓毒症心肌损伤细胞模型；通过盲肠结扎穿孔法复制脓毒症大鼠模型。利用分子生物学技术，观察该药对MasR/PI3K-Akt-mTOR通路相关位点、自噬相关指标表达的影响，证实其通过激活该通路，抑制过度自噬，改善脓毒症心肌损伤；同时应用MasR抑制剂A779、PI3K抑制剂wortmannin、mTOR抑制剂雷帕霉素拮抗相应位点，确定该药的作用靶点。研究结论如下，细胞实验部分：①芪参活血颗粒含药血清可改善脓毒症心肌细胞存活率；②该药上调MasR/PI3K-AKT-mTOR通路相关位点mRNA和蛋白表达；③下调Beclin1、ATG-5mRNA和蛋白、LC3B蛋白的表达，抑制脓毒症心肌细胞过度自噬；④其保护作用可被MasR抑制剂A779所阻断；体内实验部分：①脓毒症可导致大鼠左室收缩功能下降，芪参活血颗粒干预治疗可改善；②该药能够降低心肌损伤标志物 cTnI、BNP，改善病理损伤，减少炎症因子IL-1β、TNF-a释放；③该药在体内通过激活MasR/PI3K-AKT-mTOR通路相关位点，减少自噬相关指标表达，其作用位点位于MasR。综上所述，芪参活血颗粒通过激活MasR/PI3K-Akt-mTOR通路，抑制心肌细胞过度自噬，改善脓毒症心肌损伤，MasR为其作用靶点。此研究将为该药在临床推广应用提供理论依据。