基于Notch信号通路调控内皮祖细胞研究鹿茸对急性心梗血管内皮损伤的修复作用

Ischemic heart disease begins with vascular endothelial cells damage, cardiac repair after acute myocardial infarction also begins from vascular endothelial cells. Our preliminary work has confirmed that growing pilose antler protein has significant effect on promoting the proliferation of human umbilical vein endothelial cells, and also has a significant improvement to the cardiac structure and cardiac function of heart failure rats after myocardial infarction. Based on the TCM theory of” the kidney dominating bone and generating marrow ", there are many studies about nourishing-kidney chinese medicine pilose antler, show that abundant growth factors in pilose antler and it can promote cell proliferation and differentiation. This research will establish the cardiovascular endothelial cell damage model and rat model of acute myocardial infarction, discuss the effect of pilose antler to the proliferation, migration, and chemotaxis of cardiac vascular endothelial cell after its intervention; by identifying the number, the proliferation, adhesion and migration of endothelial progenitor cells and its ability of generating blood vessels，using the Notch signaling pathway specific blocker of DAPT and RNA interference, to explore the regulating effect of the Notch signal pathway on endothelial progenitor cells’ mobilization and differentiation to vascular endothelial cells, and confirm and evaluate growing pilose antler through regulating the Notch signal pathway to promote endothelial progenitor cells to differentiate into endothelial cells, repair vascular endothelial injury and promote angiogenesis, repair the infarction myocardial tissue, improve heart function. To find a new target for the research and development of medicine, provide the experimental basis for the traditional Chinese medicine effect of promoting and mobilizing self-repair ability and the theory of” the kidney dominating bone and generating marrow ".

急性心梗的始动及梗死后的心脏修复均从内皮细胞开始。内皮祖细胞增殖分化为内皮细胞是内皮修复、血管新生及心肌修复的关键因素，Notch信号通路是调控此过程的关键通路。课题组前期研究证实鹿茸能显著促进人脐静脉内皮细胞增殖，并能显著改善心梗后心脏结构及功能。基于中医“补肾生髓”理论和现代研究，补肾中药鹿茸有多种生长因子且有促细胞增殖分化的作用。本研究通过建立心脏血管内皮细胞损伤模型（体外实验）和急性心梗大鼠模型（体内实验），检测鹿茸干预后心脏血管内皮细胞与内皮祖细胞数量、增殖、迁移及成血管能力；采用Notch信号通路的特异性阻断剂DAPT、isRNA等方法，验证鹿茸通过调控Notch信号通路促使内皮祖细胞分化为内皮细胞，修复血管内皮、促进血管新生、修复梗死后的心肌组织，进而改善心功能的科学假说。从细胞及分子水平明确鹿茸促进及调动机体自我修复能力的机制，阐释中医“补肾生髓”理论的内涵并提供实验依据。

课题组前期已证实，鹿茸能够提高心梗后心衰大鼠的心功能，降低心脏基质胶原纤维化、延缓心室重塑，调节SERCA2a表达活性，改善心肌收缩舒张功能；此外，鹿茸的主要成分，鹿茸蛋白提取物能够促进HUVECs 的增殖。.本研究依据缺血性心脏病发病机制，一方面，先鹿茸蛋白进行蛋白质组学分析，然后构建体外缺血缺氧细胞模型，明确鹿茸蛋白对缺血缺氧损伤的心脏微血管内皮细胞和心肌细胞的修复作用，同时观察鹿茸蛋白对内皮祖细胞的调控作用。发现鹿茸蛋白通过调节PI3K/Akt 信号通路影响凋亡相关细胞因子Bcl-2、Bax 和caspase3 的表达保护受损的心脏微血管内皮细胞，同时增加细胞Notch1 信号通路相关蛋白Notch1 和Hes1 的活化及Akt/mTOR 信号通路上的Akt 和mTOR 的磷酸化水平影响内皮祖细胞活力，进而修复心脏微血管的损伤；鹿茸蛋白通过调节PI3K/Akt 信号通路影响凋亡相关细胞因子Bcl-2、Bax 和caspase3 的表达直接保护损伤的心肌细胞，减少心肌细胞凋亡。.另一方面，建立心梗大鼠动物模型，用不同浓度鹿茸粉进行干预，观察心梗后不同时间点大鼠心功能及相关指标。发现中浓度鹿茸、高浓度鹿茸可显著改善心梗大鼠心功能，减少血清BNP，减少心肌梗死面积，保护心肌细胞。高浓度鹿茸可促进心梗大鼠内皮祖细胞动员、增加循环内皮祖细胞含量，增加心梗边缘区内皮祖细胞标记物CD133含量、内皮细胞标记物CD31含量，修复血管内皮细胞损伤、促进血管新生。高浓度鹿茸可增加Notch信号通路相关蛋白Jagged1、Notch1、NICD、Hes1蛋白表达，增加Hes1、Hey2 mRNA表达，调控心梗大鼠内皮祖细胞的动员，并可能调节其分化。高浓度鹿茸还可通过调控凋亡相关细胞因子Bcl-2、Bax 和caspase3 、caspase9的表达保护损伤的心肌细胞，减少心肌细胞凋亡。.课题组对鹿茸展开多项相关研究，旨在系统挖掘鹿茸在心血管疾病治疗中的药理作用，为缺血性心脏病的治疗提供新的思路和方法。同时为补肾中药鹿茸促进及调动机体内自我修复损伤能力提供了实验数据及中医理论依据。