基于mTOR自噬信号通路、自噬相关microRNA转录调控网络探讨芪参桃红颗粒抑制心衰心肌重塑的效应机制

The prevention and treatment of heart failure(HF) is still the difficult point of cardiology. As the important pathological mechanism of HF, myocardial remodeling manifests the entire course of HF. Modern researches suggest a close connection between myocardial remodeling and cardiomyocyte autophagy, and negative regulation of microRNA on autophagy. However, no effective drugs are available for clinical selection. Qishentaohong Granule (QSTH Granule) had been proven to be effective in HF treatment. The included herbs of QSTH Granule were suggested promising effect on cardiomyocyte autophagy and myocardial remodeling by many studies. Based on related evidence, we proposed the hypothesis that " QSTH Granule may induce the beneficial and moderate cardiomyocyte autophagy by regulating the autophagy-related miRNA transcriptional regulation network, and thus play a role in inhibiting myocardial remodeling, delaying or even blocking the process of HF." To testify this hypothesis, firstly, we will duplicate the HF rat model by constricting the aortic artery to mimic the course from myocardial remodeling to HF; secondly, we will treat rat model with QSTH Granule and compare the effect with enalapril; finally, echocardiography, transmission electron microscopy, laser confocal fluorescence imaging system, miRNA high throughput sequencing techniques, bioinformatics analysis techniques, etc. will be performed to explore the true effect of different interventions on systematic, histionic, cellular and molecular level. The purposes of this program are to provide scientific evidence for the effect of QSTH Granule on myocardial remodeling and HF, and for the pharmacological mechanism of traditional Chinese medicine underling HF treatment.

心衰防治是心血管学科面临的重点和难点，心肌重塑是心衰重要的病理机制，贯穿心衰病程的始终。现代研究认为心肌细胞自噬与心衰心肌重塑密切相关，microRNA可负性调控自噬的各个阶段，但缺乏有效的干预药物可供临床选择。芪参桃红颗粒能够有效防治慢性心衰，研究证实其组成药物能够调节自噬活性、抑制心肌重塑。据此提出“芪参桃红颗粒可能通过调节自噬相关microRNA转录调控mTOR通路，介导有益并适度的心肌细胞自噬，从而起到抑制心肌重塑，延缓甚至阻断心衰进程作用”的假说。拟复制压力负荷心衰小鼠模型，模拟心肌重塑-心衰进程，使用芪参桃红颗粒进行干预，以依那普利为西药对照；运用小动物超声心动、透射电镜、激光共聚焦荧光成像系统、、质谱分析、miRNA测序、生物信息学、RT-PCR等技术，从整体、组织、细胞、分子层面展开研究。为芪参桃红颗粒防治心衰心肌重塑提供科学依据，为阐释中药防治心衰药理作用提供实验证据。

项目的背景.心肌重构是心衰发生发展的主要病理生理过程，与细胞自噬等生命活动异常变化相关。自噬过程的过度或不足都会损害心功能。目前无药物针对心肌细胞自噬机制对心肌重构发挥有效作用，探寻中医药对于心肌细胞自噬的干预影响，有重要意义。.临床心衰患者多属于中医气虚血瘀水停的病机范畴，芪参桃红颗粒在前瞻性随机对照研究中，在中医证候积分和主要症状积、左室射血分数、NT-proBNP、6分钟步行距离、患者生存质量等方面表现出有效改善，同时降低6个月内心衰再住院率，但其基础机制尚且不清。..主要研究内容.本研究以芪参桃红颗粒为干预药物，以调控自噬相关mTOR通路激动剂雷帕霉素、阻滞剂3-甲基腺嘌呤为工具药，以阳性药马来酸依那普利片为对照组，观察在药物干预下，TAC心衰小鼠不同时期心功能、心肌形态、重构及凋亡程度，自噬体形成情况，自噬相关的miRNA表达情况。..重要结果.芪参桃红颗粒可有效改善压力负荷心衰小鼠的心功能及心肌重构，其机制可能是基于mTOR信号通路调控相关miRNA，在心衰的不同时期，芪参桃红颗粒通过上调或下调心肌细胞自噬活动趋向于某种特定程度，以产生相应的保护作用。 ..关键数据及其科学意义.心功能评价：术后2周时，芪参桃红颗粒组在提高TAC心衰小鼠LVEF方面优于其他组(P<0.05)，术后4周时，芪参桃红颗粒组、依那普利组在提高LVEF方面优于其他组；同组前后比较，芪参桃红颗粒+雷帕霉素组在FS%、LVEF、LVEDd方面均有提高(P<0.05)，标准化心脏重量无增大，结果提示心功能的改善有可能是因为中药的干预，自噬水平调节至适应性水平，产生了保护心肌的作用。.各组小鼠心肌纤维总面积的定量分析证实，芪参桃红颗粒组、依那普利组在缩小TAC术后小鼠心肌总面积方面具有优势，并且，术后4周各干预组心肌总面积与中药+3-甲基腺嘌呤组相比无统计学差异，提示芪参桃红颗粒及依那普利对心肌功能的改善可能是基于增强单个心肌纤维束的舒缩能力实现的。.miRNA测序及KEGG分析结果显示，在芪参桃红颗粒组较模型组中，mTOR、Lysosome通路富集明显，与自噬密切相关，且术后2周、4周时相关miRNA表达有所差异。