基于ERs-PI3K通路的三七皂苷类成分防治心肌缺血再灌注损伤的分子机理研究

Reperfusion is the most effective treatment for acute myocardial infarction, markedly reducing mortality and morbidity. Reperfusion however induces necrotic and apoptotic damages to cardiomyocytes, that were viable prior to reperfusion, a process called Myocardial Ischemia/Reperfusion Injury (MI/RI). Over the past 30 years, hundreds of experimental interventions (both pharmacologic and nonpharmacologic) have been reported to protect the ischemic myocardium in experimental animals; however, with the exception of early reperfusion, none has been translated into clinical practice. The population-based survey assessed men have about twice the total incidence of morbidity and mortality of women, and the sex gap in morbidity tends to diminish after age 45 years. Several studies suggested that estrogen had the protective effect for MIRI, and most of researchers believed that apoptosis was the most important component for MIRI, and estrogen receptor (ER) can regulate PI3K signaling cascade, which was the most important signaling pathways involved in apoptosis. Those suggested that one of the possible mechanisms of estrogen anti MIRI was the inhibition of apoptosis by ERs-PI3K signaling cascade. Previous studies had shown that the total saponins of Pnanxnotoginseng (PNS), one of the phytoestrogen, which could regulate the PI3K signaling cascade in cell, had beneficial protective effect for MIRI. Accordingly, we propose the hypothesis that one of the probability mechanisms for the protective effects on MIRI-induced apoptosis by PNS is through ERs-PI3K signaling cascade. By establishing the rat model of myocardial MIRI, the hypoxia/reoxygenation myocardial cell model, and the gene and protein over-expression or expression inhibited myocardial cell model by transfected with Ers, PI3K, and eNOS inhibitor, the intervention effects of PNS is observed at molecular, cellular, and animal levels. This study could provide a new approach and theoretical basis for phytoestrogen composition in prevention and treatment of MIRI.

心肌缺血再灌注损伤（MIRI）已成为临床冠心病患者获得再灌注治疗最佳疗效的主要障碍，细胞凋亡是MIRI产生的关键环节，PI3K通路是参与细胞凋亡最重要的信号通路，雌激素受体（ERs）激活后可通过PI3K通路调控下游通路达到治疗多种疾病的目的。前期研究表明具有雌激素样活性的三七皂苷类成分具有很好的抗MIRI作用，课题组前期已证实三七皂苷类成分能够调控ERs-PI3K通路及下游通路抑制细胞凋亡，由此提出假说：三七皂苷通过调控ERs-PI3K通路抑制细胞凋亡保护MIRI。为验证该假说，本项目拟通过多种生物学技术考察PNS对MIRI模型ERs-PI3K通路中ER、p85α、Ras、AKt等关键蛋白、基因表达的影响，并应用靶点拮抗剂进行“功能缺失”验证，旨在从分子层面阐明三七皂苷抗MIRI的机理，为其深入开发提供理论依据，另一方面也丰富了MIRI的致病理论，为确认MIRI新的治疗靶点提供参考。

心肌缺血再灌注损伤是目前影响冠心病获得再灌注最佳疗效的最主要障碍，目前还没有有效的药物进行治疗。前期研究发现三七总皂苷在对心肌缺血再灌注损伤具有较好的治疗作用，但其作用机制还不清晰。本研究在课题组前期研究的基础上对三七总皂苷防治心肌缺血再灌注损伤的药效物质基础及作用机制进行了深入研究。结果发现，三七总皂苷在80-40mg/kg/d的剂量范围内给药5d，能够对心肌缺血再灌注损伤损伤模型大鼠的心脏组织具有很好的保护作用，并对心脏组织中雌激素受体蛋白及心肌细胞凋亡蛋白的表达量影响显著，但对正常未成年雌鼠的血清中雌激素水平、子宫系数及卵巢系数并未有显著性影响。随后我们通过UPLC-MS方法测定了三七总皂苷的入血成分，并通过主成分分析及判别分析等方法筛选得到17个引起体内次生代谢产物差异较大的化合物，通过查阅文献及数据库检索，鉴定了15个化合物结构，证实了三七皂苷R1及人参皂苷Rg1对大鼠体内次生代谢产物影响最大，并利用体外模型证实了25μg/mL剂量下三七皂苷R1对缺氧/复氧处理后H9c2心肌细胞的保护作用要优于PNS及人参皂苷Rg1，以此为基础，利用雌激素受体抑制剂及PI3K抑制剂深入研究了三七皂苷R1作用机制，结果证实三七皂苷防治心肌缺血再灌注损伤的作用机制之一就是能够通过影响雌激素受体α蛋白表达，调控PI3K/p-Akt通路以及其下游细胞凋亡通路蛋白，最终发挥保护受损心肌的作用。本研究进一步阐明了三七总皂苷防治心肌缺血再灌注损伤的药效物质基础，为三七总皂苷产品开发及应用奠定了理论基础，同时丰富了心肌缺血再灌注损伤的防治理论的丰富并寻找到潜在的治疗靶点，也为同类药物的开发提供了借鉴。