通脉养心丸通过上调GPER激活HIF-1α/eNOS信号通路减轻心肌缺血再灌后无复流的作用机制研究

Mechanism of Tongmai Yangxin pill to reduce the no-reflow after myocardial ischemia and reperfusion by activating HIF-1α/eNOS signaling pathway up-regulated by GPER

基于整合药理学及实验验证探讨通脉养心丸(Tongmai Yangxin pill,TMYX)对心肌缺血再灌后无复流(no-reflow,NR)的心脏保护作用及其分子机制。首先采用心肌NR大鼠(心肌缺血2 h后再灌注2 h)证实TMYX减轻NR的作用;再通过整合药理学分析、NR大鼠离体冠脉微血管体外研究和NR大鼠体内研究,揭示TMYX改善NR的主要成分、靶点和途径。本实验获得湖南中医药大学伦理委员会批准(LLBH-202212160001)。结果发现,TMYX通过改善心脏结构和功能,减少无复流、缺血心肌面积和心肌细胞病理损伤,降低心肌肌钙蛋白I(cardiac troponin I,cTnI)含量,对NR有治疗作用。此外,整合药理学预测TMYX改善NR机制与缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)、核因子κB(nuclear factor kappa-B,NF-κB)和肿瘤坏死因子(tumor necrosis factor,TNF)信号通路有关。在体内,TMYX增加G蛋白偶联雌激素受体(G protein-coupled estrogen receptor,GPER)、磷酸化细胞外信号调节激酶(phospho-extracellular signal-regulated kinase,p-ERK)和HIF-1α的表达。在体外,TMYX增强冠脉微血管的舒张功能,然而此作用被GPER、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及一氧化氮受体(soluble guanylate cyclase,sGC)阻断剂抑制。本研究通过整合药理学和实验评估,揭示TMYX通过上调GPER激活HIF-1α/eNOS信号通路,舒张冠状动脉微血管,从而显著改善NR。

The Tongmai Yangxin pill(TMYX)has potential clinical effects on no-reflow(NR);however,the effective substances and mechanisms by which this occurs remain unclear.This study evaluates the cardioprotective effects and molecular mechanisms of TMYX against NR.We used a myocardial NR rat model(2 h after myocardial ischemia and 2 h after reperfusion)to confirm the effect and mechanism of action of TMYX in alleviating NR.In vitro studies in isolated coronary microvasculature of NR rats and in silico network pharmacology analyses were performed to reveal the underlying mechanisms of TMYX and determine the main components,targets,and pathways of TMYX,respectively.The experiment was approved by the Ethics Committee of Hunan University of Chinese Medicine(LLBH-202212160001).TMYX showed therapeutic effects on NR by improving cardiac structure and function,reducing NR,ischemic areas,and cardiomyocyte injury,and decreasing the content of cardiac troponin I(cTnI).Moreover,the mechanism of TMYX predicted by network pharmacology is related to the hypoxia inducible factor-1(HIF-1),nuclear factor kappa-B(NF-κB),and tumor necrosis factor(TNF)signaling pathways.TMYX increased the expression of G protein-coupled estrogen receptor(GPER),phospho-extracellular signal-regulated kinase(p-ERK),and HIF-1α.In vitro,TMYX enhanced the diastolic function of coronary microvascular cells;however,this effect was inhibited by GPER inhibitor(G-15),eNOS inhibitor(L-NAME),and sGC inhibitor(ODQ).This study integrates pharmacology and experimental evaluation to reveal that TMYX activates HIF-1α/eNOS signaling pathway by upregulating GPER to relax coronary microvessels,thereby significantly alleviating NR.