线粒体动力学相关MicroRNA在气虚血瘀病症结合大鼠模型中的意义及益气活血中药干预机制研究

In traditional Chinese medicine(TCM), qi deficiency and blood stasis is regarded as the main pathogenesis of ischemic heart diseases. While in western medicine, it shows that mitochondrial dynamics is closely related to ischemic heart diseases. But the relationship between qi deficiency and blood stasis and mitochondrial dynamics is still unclear yet. Previous researches confirm that MicroRNA, which is associated with mitochondrial dynamics, expresses differently in patients with ischemic heart diseases in qi deficiency and blood stasis syndrome. And Shuangshenningxin prescription can prevent ischemic myocardial injury by improving energy metabolism. Basing on these, we propose that the abnormal expression of mitochondrial dynamics associated MicroRNA is a potential mechanism in ischemic heart diseases, and Shuangshenningxin prescription can maintain the stability of mitochondrial dynamics by regulating associated MicroRNA, so as to treat ischemic heart diseases in qi deficiency and blood stasis syndrome. In this study, we will take it as a breakthrough point. On the on hand, animal experiments will be carried out to confirm the different expression of mitochondrial dynamics associated MicroRNA in animal models with qi deficiency and blood stasis syndrome, and discovery the regulatory effect of Shuangshenningxin prescription on associated MicroRNA. On the other hand, cell experiments will be carried out to discover the regulatory mechanism of Shuangshenningxin prescription on associated MicroRNA. All the outcomes will contribute to the declaration of the regulatory effect of Shuangshenningxin prescription on MicroRNA- mitochondrial dynamics- qi deficiency and blood stasis syndrome chain, so as to provide new evidences for the theoretical researches on qi deficiency and blood stasis in TCM.

中医认为气虚血瘀是缺血性心脏病的主要病机，西医发现缺血性心脏病与线粒体动力学关系密切，然而气虚血瘀与线粒体动力学之间关系仍不明确。前期研究证实线粒体动力学相关MicroRNA在气虚血瘀型缺血性心脏病患者血液内有差异表达，双参宁心方可通过保护线粒体改善能量代谢防治缺血导致的心肌损伤。据此我们认为线粒体动力学相关MicroRNA的异常表达是气虚血瘀型缺血性心脏病的潜在机制，双参宁心方可通过调节相关MicroRNA，维持线粒体动力学稳定，发挥对气虚血瘀型缺血性心脏病的治疗作用。本研究将以此为切入点，通过动物实验验证线粒体动力学相关MicroRNA在气虚血瘀动物模型中的差异表达及药物对相关MicroRNA的调节作用，通过细胞模型考察药物对相关MicroRNA的调节机制，以阐明双参宁心方对“MicroRNA-线粒体动力学-气虚血瘀”作用链的调控作用，为中医气虚血瘀传统理论研究提供新的依据。

前期研究发现线粒体动力学相关MicroRNA在气虚血瘀型缺血性心脏病患者血液内有差异表达，双参宁心方可通过保护线粒体改善能量代谢防治缺血导致的心肌损伤，再次研究背景下提出假说：线粒体动力学相关MicroRNA的异常表达是气虚血瘀型缺血性心脏病的潜在机制，益气活血中药复方可通过调节相关MicroRNA，维持线粒体动力学稳定，发挥对气虚血瘀型缺血性心脏病的治疗作用。本项目采用睡眠剥夺复合冠状动脉左前降支结扎的方法建立在体大鼠冠心病气虚血瘀证病证结合动物模型，考察线粒体动力学相关MicroRNA的异常表达情况，以及益气活血中药复方对模型大鼠心肌损伤的保护作用，及相关MicroRNA的调控作用。研究结果表明模型动物与正常动物比较，多种线粒体动力学相关的MicroRNA表达具有显著差异，对相关的microRNA进行QPCR定量分析发现，Rno-Mir-139-5p、Rno-Mir-345-3p、Rno-Mir-505-3p、Rno-Mir-31a四个microRNA表达量变化明显，同时药物对以上四个microRNA具有调节作用。同时发现益气活血方对气虚血瘀心脏病大鼠模型具有显著的心脏保护作用，主要表现在改善模型大鼠的左室射血分数和短轴缩短率，同时对中医证候学指标舌像、脉搏幅度及抓力等也具有显著的改善作用。病理学结果亦证实益气活血方对模型大鼠的保护作用，显著减轻缺血导致的炎性细胞浸润及纤维化程度等对线粒体超微结构也具有改善作用。机制研究发现益气活血方显著增加模型大鼠血清中NT-proBNP、ALD、AngⅡ和ATP含量，发挥心肌保护的作用。模型组大鼠心肌组织内PINK1、Pakin、caspase3蛋白含量明显升高，给药组对以上三个蛋白具有明显的抑制作用。PINK1 / Parkin途径是介导线粒体自噬的机制之一，与线粒体分裂融合直接相关，结果提示益气活血方可通过调控线粒体动力学相关microRNA，从而改善线粒体功能，影响心肌能量代谢水平，从而发挥心肌保护的作用。该项目进一步明确了益气活血方的心肌保护作用，明确其通过线粒体途径影响能量代谢水平，对气虚血瘀型冠心病大鼠具有很好的改善作用，效果显著机理清楚，具有很好的临床应用前景。