芒柄花黄素通过上调GPER激活PI3K/Akt/e NOS信号通路减轻心肌缺血再灌后无复流的作用及机制研究

The effect and mechanism of formononetin on alleviating no-reflow after myocardial ischemia and reperfusion by up-regulating the PI3K/Akt/eNOS signal pathway activated by GPER

基于整合药理学及实验验证探讨芒柄花黄素(formononetin,FMN)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,采用人乳腺癌细胞(MCF-7)和心肌NR大鼠证实FMN的雌激素活性和减轻NR的药效作用。将雄性Sprague-Dawley (SD)大鼠分为Sham、NR、FMN (20 mg·kg^(-1))和硝普钠(sodium nitroprusside,SNP,5.0 mg·kg^(-1))组,给药1周,每天1次,本实验获得天津中医药大学伦理委员会批准(TCM-LAEC2019095)。再整合药理学分析和NR大鼠体内研究,揭示FMN改善NR的作用机制。结果发现,FMN具有雌激素样作用,且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,起到减轻NR的作用。整合药理学预测FMN改善NR机制主要与磷脂酰肌醇-3-激酶-蛋白激酶B (phosphatidyinositol-3-kinase-protein kinase B,PI3K-Akt)信号通路有关。植物雌激素主要通过上调G蛋白偶联雌激素受体(G protein-coupled estrogen receptor,GPER)发挥保护心血管的作用,GPER也作为PI3K-Akt信号通路上游的重要调节因子,本研究发现FMN能显著上调GPER、p-PI3K、p-Akt和磷酸化内皮型一氧化氮合酶(phospho-endothelial nitric oxide synthase,p-eNOS)蛋白表达,且与GPER及eNOS蛋白具有良好结合能力。本研究通过整合药理学和实验评估,揭示FMN通过上调GPER激活PI3K/Akt/eNOS信号通路,从而显著改善NR的机制。

To investigate the cardioprotective effect of formononetin(FMN)on no-reflow(NR)after myocardial ischemia-reperfusion and its molecular mechanism based on integrated pharmacology and experimental verification,firstly,human breast cancer MCF-7 cells and myocardial NR rats were used to confirm the estrogenic activity and the effect of alleviating NR of FMN,respectively.Male SD rats were divided into Sham,NR,FMN(20 mg·kg^(-1))and sodium nitroprusside(SNP,5.0 mg·kg^(-1))groups,which were administered once a day for one week,the experiment was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine(TCM-LAEC2019095).The pharmacological analysis and in vivo study of NR rats were integrated to reveal the mechanism of FMN improving NR.The results showed that FMN had estrogenic effect and reduced NR by improving cardiac structure and function,reducing NR,ischemic myocardial area and pathological injury of cardiomyocytes.Integrated pharmacology predicts that the mechanism of FMN improving NR is mainly related to phosphatidyinositol-3-kinase-protein kinase B(PI3K-Akt)signal pathway.Phytoestrogens play a role in cardiovascular protection mainly by activating G protein-coupled estrogen receptor(GPER).GPER is also an important regulator in the upstream of PI3K-Akt signaling pathway.This study found that FMN can significantly activate GPER,p-PI3K,p-Akt and phospho-endothelial nitric oxide synthase(p-eNOS).It has good binding ability with GPER and eNOS protein.In this study,through the integration of pharmacology and experimental evaluation,it is revealed that FMN activates PI3K/Akt/eNOS signal pathway by activating GPER,thus significantly improving NR.