从FXR/SHP通路探讨电针预处理减轻大鼠心肌缺血/再灌注损伤的线粒体机制研究

Cardiovascular disease ranks among the top in the current three deadly diseases, myocardial ischemia reperfusion injury (MIRI) is a serious clinical phenomenon, and how to reduce the MIRI has become the focus of current events. Acupuncture preconditioning can reduce MIRI, though its mechanism is not fully clear, it is closely related to mitochondria. FXR/SHP is a latest finding pathway which is related to MIRI. Though it can activate the mitochondrial apoptosis pathway and leads to cardiomyocyte apoptosis. The clinical effect of acupuncture on myocardial ischemia is significant, so far, there has been no report about acupuncture mediate the FXR/SHP pathway of the relationship between MIRI and apoptosis. On the basis of the previous study, we used the modern molecular biology techniques, such as gene silencing, qPCR and so on. By comparing the change of MIRI group, electroacupuncture pretreatment group, FXR silence group, electroacupuncture pretreatment plusing FXR silent group of myocardial mitochondrial apoptosis related genes and FXR SHP genes. To observe the effect of electroacupuncture pretreatment and FXR silencing on the expression of related genes, We will further elucidate the mitochondrial mechanism of electroacupuncture preconditioning on MIRI in rats by regulating mitochondrial apoptosis in FXR/SHP pathway, to provide the basis for clinical diagnosis and treatment of clinical optimization for the prevention and cure of MIRI.

心血管疾病位居三大致死疾病的榜首，心肌缺血再灌注损伤（MIRI）是一个很严重的临床现象，如何降低MIRI已成为目前的研究重点。针刺预处理可减轻MIRI，但其机制尚未明确。研究表明，MIRI与线粒体密切相关，FXR/SHP是最新发现的一条与MIRI相关的通路，能激活线粒体凋亡途径引起心肌细胞凋亡，目前尚无针灸调控FXR/SHP通路与MIRI的相关报道。我们在前期研究的工作基础上，以心肌缺血再灌注大鼠为研究对象，应用基因沉默、qPCR等现代分子生物学技术，通过比较MIRI组、电针预处理组、FXR沉默组、电针预处理+FXR沉默组心肌线粒体凋亡相关基因及FXR、SHP基因的变化，观察电针预处理及FXR沉默对相关基因表达的影响，拟从FXR/SHP通路调控线粒体凋亡阐明电针预处理减轻大鼠MIRI的作用机制，为电针预处理防治MIRI及临床优化临床诊疗方案提供依据。

选用MIRI大鼠为研究对象，在中医针灸“治未病”理论指导下，进行电针预处理、FXR抑制剂及FXR基因沉默干预，通过NBT染色法、ELISA法、荧光定量PCR（qRT-PCR）、基因沉默等实验方法，观察心肌损伤相关指标、线粒体功能相关指标、心肌线粒体凋亡相关基因及FXR/SHP通路相关基因表达的变化情况。结果表明电针预处理能明显降低MIRI大鼠心肌梗死面积及梗死区质量、降低血清LDH、CK、CK-MB和cTnI含量、改善大鼠心功能，减轻心肌细胞损伤；电针预处理可以有效抑制线粒体mPTP开放，提高心肌细胞中ATP的含量、降低ADP、AMP的含量，降低ROS的生成和膜电位水平，激活心肌线粒体呼吸链复合物酶活性，激活线粒体Na--K＋-ATP酶、Ca2＋-Mg2＋-ATP酶活性，保护心肌线粒体，改善MIRI；电针预处理可以显著降低心肌缺血再灌注损伤过程中FXR、SHP、Cyt-c、Caspase-9、Caspase-3等基因表达水平，说明电针预处理通过调控FXR/SHP通路介导线粒体凋亡通路，以抑制心肌细胞凋亡，改善MIRI。与电针组相比，AAV-shRNA-FXR通过对FXR基因进行沉默，可进一步加强电针预处理对MIRI大鼠的心肌细胞凋亡水平、线粒体凋亡因子（Cyt-c、Caspase-9、Caspase-3）及FXR/SHP通路（FXR、SHP）的mRNA表达水平的抑制效果，表明电针预处理通过FXR/SHP通路介导的线粒体凋亡发挥MIRI 。本研究为临床防治MIRI提供了新思路和科学依据，对临床诊治有十分重要的理论意义和实用价值。