miR-1调控Bcl-2/Bax介导的益气活血方抗心肌细胞凋亡研究

Myocardial cell apoptosis is the basic pathological process during myocardial ischemia reperfusion injury (MIRI) after acute myocardial infarction. It’s known that microRNA-1 (miR-1) has a negative regulatory effect on myocardial cell apoptosis, while the mechanism is not clear. Based on rat model of MIRI and H9c2 cell model of hypoxia/reoxygenation (H/R), the present project is trying to determine the correlation of cardiac apoptosis with overexpression of miR-1 or inhibition of its expression through genetic modification. We also explore the mechanism underlying cardiac apoptosis by measuring expression of miR-1 and its targeted genes, acquiring mitochondrial function, and conducting calcium signal. Finally, Yiqihuoxue Decoction is applied in advance to pre-treat MIRI rats and H/R cells, and the effects of Yiqihuoxue Decoction are conducted on the expression of miR-1, Bcl-2/Bax, mPTP, Cyt C, and caspase-3, as well as calcium signal, to investigate the protective effects of Yiqihuoxue Decoction on cardiac apoptosis. This project will help elucidate the mechanism of Yiqihuoxue Decoction against myocardial cell apoptosis and provide a theoretical basis for clinical medication.

急性心梗后心肌缺血再灌注损伤（MIRI）病理基础为心肌细胞凋亡，微小RNA-1（miR-1）对心肌细胞凋亡具有负性调控作用；前期研究证实益气活血方具有抑制心梗后左室重构大鼠心肌细胞凋亡的作用，但机制不明。本项目利用大鼠MIRI模型和心肌细胞H9c2缺氧/复氧（H/R）模型，通过基因修饰方法对miR-1过表达和表达抑制来明确其与心肌细胞凋亡的相关性；通过研究MIRI及H/R后miR-1异常表达及其靶基因表达差异、测定心肌细胞线粒体膜功能状态以及钙信号变化等，探讨miR-1调控心肌细胞凋亡的分子机制；观察益气活血方预处理后MIRI及H/R条件下miR-1、Bcl-2/Bax、mPTP、Cyt C、caspase-3、Ca2+水平变化，探讨益气活血方在心肌细胞凋亡发生发展过程中的保护作用。本项目的完成将有助于阐明益气活血方抗心肌细胞凋亡的作用机制，为临床用药提供理论支持。

本项目研究益气活血方急性心梗后心肌缺血再灌注损伤（MIRI）细胞凋亡的干预效应及作用机制。制备了大鼠MIRI模型、H9c2心肌细胞缺氧复氧损伤（H/R）模型以及大鼠隔离心肌细胞H/R模型，结果表明益气活血方及其单体能明显减轻MIRI，减轻细胞凋亡程度（Bax、caspase-3水平降低，Bcl-2水平升高），稳定线粒体膜电位及细胞钙离子浓度；益气活血方也能降低促栓因子PAI-1、TF表达水平，降低NF-kB炎性通路活性，升高解偶联蛋白2（UCP2）以及核转录因子KLF2表达水平；益气活血方还能降低细胞与组织miR-1表达水平。利用NF-kB通路抑制剂、KLF2沉默和过表达修饰，发现益气活血方通过抑制NF-kB活性、升高KLF2及UCP2表达水平发挥抗心肌细胞损伤、凋亡以及减轻血栓形成作用。利用miR-1模拟物和抑制物、IGF-1沉默和过表达工具，以及双荧光素酶实验，阐明了miR-1通过抑制Bcl-w和IGF-1水平促进H/R诱导的心肌细胞凋亡。本研究还发现了红花单体羟基红花黄色素A、丹参花提取物DHS-20通过调控miR-1发挥抗氧化损伤及凋亡作用。本项目的完成，阐明了miR-1对MIRI过程中细胞凋亡的调控作用及益气活血方的干预效应机制，具有很好的创新性和实际意义，为临床治疗MIRI用药提供了确切的理论依据。