基于内皮祖细胞外泌体miR-126探讨温心方保护动脉粥样硬化血管内皮的机制研究

Dysfunction and damage of blood vessel endotheliocytes is the trigger of atherosclerosis, which is crucial pathological basic of coronary heart disease. From our preliminary study, we demonstrated that Wen-Xin-Formula could promote the expression of eNOS by stimulating the PI3K/Akt pathway, which could alleviate the damage of blood vessel endotheliocytes in atherosclerosis. Recent research has suggested that exosomes secreted by endothelial progenitor cells which contain plenty of miR-126 are playing an essential role in the stimulation of PI3K/eNOS/NO pathway. According to our preliminary study, we hypothesize that Wen-Xin-Formula could promote the repairing of the damaged blood vessel endotheliocytes by stimulating the PI3K/Akt pathway through regulation of miR-126 in exsomes released by endothelial progenitor cells. In this research, we will establish atherosclerotic models in vivo by ApoE knockout mice fed with high fat diet and ex vivo adopting damaged endotheliocytes and endothelial progenitor cells induced by ox-LDL. Concetrating on miR-126 and PI3K/Akt pathway, we will also apply miRNA interference technology, Western Blot and qRT-PCR to explore the mechanism of Wen-Xin-Formula protecting against dysfunction of blood vessel endotheliocytes in atherosclerosis, in order to suply theoretical and experimental basics for Wen-Xin-Formula to cure atherosclerosis clinically.

动脉粥样硬化是冠心病的重要病理基础，血管内皮损伤和功能异常是动脉粥样硬化发生的始动环节。前期研究证实温心方能激活PI3K/Akt信号通路促进eNOS表达，减轻动脉粥样硬化血管内皮损伤；最新研究证实内皮祖细胞分泌的外泌体中富含的miR-126是激活内皮细胞PI3K/eNOS/NO信号通路的关键。结合前期基础，我们提出假说：温心方可通过调控内皮祖细胞外泌体miR-126激活内皮细胞PI3K/Akt信号通路减轻动脉粥样硬化血管内皮损伤。本研究采用高脂喂养ApoE-/-基因敲除小鼠和氧化型低密度脂蛋白诱导内皮细胞、内皮祖细胞损伤建立体内外动脉粥样硬化模型，应用miRNA干扰技术、qRT-PCR、Western Blot等方法，紧密围绕内皮祖细胞外泌体miR-126这一靶点及PI3K/Akt信号通路，探讨温心方保护动脉粥样硬化血管内皮的关键机制，为温心方治疗动脉粥样硬化提供科学依据。

项目背景: 动脉粥样硬化是冠心病的重要病理基础，血管内皮损伤和功能异常是动脉粥样硬化发生的始动环节。前期研究证实温心方能激活PI3K/Akt信号通路促进eNOS表达，减轻动脉粥样硬化血管内皮损伤；最新研究证实内皮祖细胞分泌的外泌体中富含的miR-126是激活内皮细胞PI3K/eNOS/NO信号通路的关键。结合前期基础，我们提出假说：温心方可通过调控内皮祖细胞外泌体miR-126激活内皮细胞PI3K/Akt信号通路减轻动脉粥样硬化血管内皮损伤。 .主要研究内容：本研究采用高脂喂养ApoE-/-基因敲除小鼠和氧化型低密度脂蛋白诱导内皮细胞、内皮祖细胞损伤建立体内外动脉粥样硬化模型，应用miRNA干扰技术、qRT-PCR、Western Blot等方法，紧密围绕内皮祖细胞外泌体miR-126这一靶点及PI3K/Akt信号通路，探讨温心方保护动脉粥样硬化血管内皮的关键机制，为温心方治疗动脉粥样硬化提供科学依据。.重要结果：①温心方及经温心方修饰的外泌体能显著调脂降脂，并降低引起和血管内皮功能紊乱的危险因素、表达，对Apoe-/-动脉粥样硬化模型小鼠内皮具有显著的保护作用；②温心方可通过调控内皮祖细胞外泌体miR-126激活内皮细胞PI3K/Akt信号通路减轻动脉粥样硬化血管内皮损伤。.关键数据：温心方能够上调氧化型低密度脂蛋白诱导的内皮祖细胞外泌体miR-126表达；经温心方修饰的外泌体能够在生理范围内升高ox-LDL诱导的内皮细胞中及动脉粥样硬化小鼠血清NO含量，能升高NO/ET-1，降低Ang-II的表达；经温心方修饰的EPCs外泌体能通过miR126上调PI3KR2蛋白表达，上调PI3Kp85、eNOS蛋白表达，能够增强AKT及eNOS蛋白的磷酸化，增加AKT及eNOS的利用率，从而上调PI3K/AKT/eNOS通路的表达；温心方及温心方修饰的外泌体能有效改善Apoe-/-动脉粥样硬化小鼠的血脂水平，降低LDL、CHO、TG含量，改善小鼠主动脉根部斑块面积。 .科学意义：明确温心方对动脉粥样硬化具有血管内皮保护作用，为中医药防治动脉粥样硬化提供实验依据，为今后动脉粥样硬化患者的临床治疗提供一定的借鉴。