香青兰总黄酮调控AMPK/SIRT1/PGC-1α信号通路抗心肌缺血再灌注损伤作用机制的研究

Myocardial ischemia reperfusion injury（MIRI）is a common cause of sudden death in clinical patients with heart diseases. Mitochondrial dysfunction is one of the important mechanisms of MIRI. A new important discovered intracellular signaling pathway named a regulating cell energy output signal network which constitutes AMPK, SIRT1and PGC-1ɑ play an important role in regulating mitochondrial energy metabolism, resistance to oxidative stress and improve the function of mitochondria. Dracocephalum Moldavica total flavones (TFDM) is a kind of natural flavonoids obtained from the characteristic advantage resources Dracocephalum Moldavica in Xinjiang and it had a clear effect against myocardial ischemia and its mechanism against MIRI might be closely related to regulating AMPK/SIRT1/PGC-1ɑ signal transduction pathway according to our before research. Based on preliminary findings the new methods and technologies of modern pharmacological research will be used by establishing the models of neonate rats’ myocardial and rats’ myocardial ischemia reperfusion injury, and the influence on the development of MIRI with TFDM will start from the AMPK/SIRT1/PGC-1ɑ signaling pathway. New targets and mechanism of TFDM against MIRI from the level of overall, cells, protein and molecular will be clarified to establish a theoretical basis for further exploitation and utilization of this traditional Uighur genuine medicine. The results of the research will have certain guiding significance to promote and facilitate the study of Dracocephalum Moldavica modernization.

心肌缺血再灌注损伤（MIRI）是临床心脏病患者猝死的常见原因。线粒体功能异常是引起MIRI的重要机制之一。AMPK、SIRT1和PGC-1ɑ构成一个调节细胞能量输出的信号网络，是近年来新发现的一条重要的细胞内信号通路，在调节线粒体能量代谢、抗氧化应激和改善线粒体功能方面发挥重要作用。香青兰总黄酮（TFDM）是从新疆特色资源香青兰中提取的一类天然黄酮类活性成分。前期研究显示，TFDM抗心肌缺血作用确切，其机制可能与调控AMPK/SIRT1/PGC-1ɑ信号转导通路密切相关。因此，本研究以AMPK/SIRT1/PGC-1ɑ信号通路为切入点，通过大鼠心肌缺血再灌注损伤及心肌细胞缺氧/复氧损伤模型，探讨TFDM调控AMPK/SIRT1/PGC-1ɑ信号通路对MIRI发生发展的影响，为TFDM抗MIRI提供理论和实践依据。研究结果对促进维药香青兰现代化研究和推动维药作用机理研究具有一定的指导意义。

通过结扎大鼠心脏左冠状动脉前降支，缺血30min后，再灌注2h，构建I/R模型。采用H9C2细胞缺氧2h后复氧4 h，建立H/R损伤模型。应用 TFDM、AMPK抑制剂Compound C、SIRT1抑制剂EX-527进行干预。心肌梗死面积检测及心肌组织病理学形态观察；采用试剂盒检测心肌组织活性氧（ROS）、超氧化物歧化酶（SOD）、丙二醛（MDA）含量及ATP、ADP、AMP和NAD+含量，透射电子显微镜观察心肌线粒体超微结构。实时荧光定量RT-PCR、免疫组化及 Western Blot 检测 AMPK、SIRT1及PGC-1α 基因及蛋白的表达水平。在细胞水平上，WB法检测AMPK、SIRT1、PGC-1α蛋白表达水平；实时荧光定量RT-PCR、免疫组化及 WB法检测 AMPK、SIRT1及PGC-1α 基因及蛋白的表达水平。结果，动物实验结果表明：TFDM预处理后，TFDM组心肌梗死面积明显减少；显降低缺血再灌注大鼠血清中ROS水平，提高SOD活性；降低MDA含量；可以调节MIRI后的能量代谢障碍，TFDM组线粒体损伤程度明显减轻。上调AMPK、SIRT1及PGC-1α m RNA的表达，与MIRI组比较差异存在统计学意义；TFDM组SIRT1及PGC-1α蛋白的表达显著增加。细胞实验表明：改善了I/R大鼠心肌病理学形态；显著提高H/R后H9C2细胞的活力；明显提高线粒体呼吸链复合物 Ⅰ、Ⅲ、Ⅳ、Ⅴ 的活力。TFDM组SIRT1及PGC-1α蛋白表达显著增加，p-AMPK蛋白表达也进一步增多。研究阐明了TFMD 调控AMPK/SIRT1/ PGC-1α信号通路的心肌保护的靶向机制，为维药香青兰在临床心肌缺血的治疗及其进一步深入开发提供科学依据，为进一步发掘利用这一传统的维吾尔道地药材奠定基础。研究工作对于香青兰创新药物的研发具有重要的指导意义，对于稳定和建设新疆新药研发人才队伍也具有重要的现实意义。