基于外泌体调控的黄芪甲苷发挥治疗性血管新生作用的机制研究

The therapeutic angiogenesis is a research hotspot for the treatment of ischemic heart disease in recent years. It is believed that angiogenesis in ischemic region of myocardium may lead to the construction of new collateral circulation and resumption of blood supply, and eventually improve the patient's symptoms. Previous study found that astragaloside Ⅳ, the representative compound of Astragalus membranaceus (Fisch.) Bungecan, could regulate the secretion of exosomes derived from myocardial cell, thereby inducing proliferation and migration of human umbilical vein endothelial cells (HUVEC) under hypoxic conditions, and the induction of exosomes was stronger than astragaloside Ⅳ, indicating that exosomes could be a bridge connecting myocardial cells and endothelial cells in cardiac microenvironment. However, there have been few studies on the role of exosomes in the field of therapeutic angiogenesis. This research intends to carry out the study on the mechanism of astragaloside Ⅳ in the field of therapeutic angiogenesis which is regulated by exosomes, including the differential proteomics on exosomes and the studies on related signaling pathways, such as MAPK pathway and PI3K/AKT pathway. The aim of this study is to investigate how does the exosomes, derived from myocardial cell which is treated by astragaloside Ⅳ, efficiently exert integrated regulating effect on angiogenesis. This research will clarify the mechanism of therapeutic angiogenesis from a new point of view, and provide new ideas for further research.

治疗性血管新生是近年来缺血性心脏病治疗的研究热点，通过促进心肌缺血区域新血管的生成，开放新的侧支循环通路，最终达到恢复缺血区域血流供应、改善患者症状的目的。本小组前期研究发现，黄芪甲苷可调控心肌细胞来源外泌体（exosomes）的分泌，从而诱导缺氧环境下人脐静脉内皮细胞增殖和迁移，且该诱导作用优于黄芪甲苷直接干预组，提示exosomes在由心肌和内皮细胞组成的心脏微环境中起关键桥梁作用。然而，目前鲜有关于exosomes在治疗性血管新生领域发挥该桥梁作用的系统研究。本课题拟开展基于exosomes调控的黄芪甲苷发挥治疗性血管新生作用的机制研究，开展exosomes差异蛋白质组学研究，探究黄芪甲苷通过调控exosomes的分泌多靶点整合调控MAPK通路和PI3K/AKT通路从而发挥其高效的促血管新生作用的机制。本课题将从新视角阐明治疗性血管新生的作用机制，为后续转化研究提供新的研究思路。

治疗性血管新生是近年来缺血性心脏病治疗的研究热点，通过促进心肌缺血区域新血管的生成，开放新的侧支循环通路，最终达到恢复缺血区域血流供应、改善患者症状的目的。本研究通过体内建立大鼠心肌缺血模型，观察不同处理组心肌细胞来源的exosomes促血管新生及抗心肌缺血损伤的作用，发现黄芪甲苷通过促进心肌细胞分泌exosomes作用于心肌缺血大鼠，减少心肌缺血大鼠梗死面积，减少缺血损伤，增强坑氧化能力，促进血管生成。体外建立HUVEC缺氧模型，采用CCK-8法、Transwell法以及Matrigel管腔形成实验考察各组心肌细胞来源的exosomes及黄芪甲苷对缺氧环境下内皮细胞存活率、细胞迁移和管腔形成的影响，发现黄芪甲苷通过促进心肌细胞分泌exosomes增加缺氧HUVEC存活率，促进细胞迁移和管腔形成。采用Western blot法检测exosomes作用后的内皮细胞中ERK、p-ERK、JNK、p-JNK、p38、p-p38、bax、bcl-2、AKT、p-AKT、VEGF的蛋白水平，发现黄芪甲苷可通过调控心肌细胞来源exosomes的分泌多靶点整合调控MAPK通路和PI3K/AKT通路从而抑制HUVEC细胞凋亡，促进血管生成。本研究表明，黄芪甲苷可通过调控心肌细胞来源exosomes的分泌，多靶点整合调控MAPK通路和PI3K/AKT通路，从而发挥其促血管新生的作用，为缺血性心脏病治疗性血管新生领域的临床治疗提供新的科学依据和研究思路。