祛瘀生新法干预Sirt1与心脏干细胞改善心肌梗死预后功能的机制研究

The prognosis of myocardial infarction (MI) is primarily affected by the pathogenesis of dysfunction of injured myocardium. How to improve myocardial cells regeneration or maintain their function are the key to promote comprehensive treatment effect. Silent information regulator 1 (Sirt1) requires nicotinamide-adenine dinucleotide (NAD) for its deacetylase activity. c-kit-positive/Lin-negative is an important marker of cardiac stem cells (CSC). Currently, many research evidence demostrates Sirt1 could induce resistance to oxidative stress and apoptosis and extend animal lifespan. Cardiac stem cells reside in the heart and constantly give rise to myocyte progenies that offer novel therapeutic options for the management of this devastating disease. The integrated traditional Chinese medicine (TCM) treatment on myocardial infarction is effective and plays an important role in China. On the short-tern explored support project of National Natural Science Foundation of China (NSFC), we discovered the use of removing blood stasis for regeneration method in traditional Chinese medicine could regulate Sirt1 signal to inhibit myocardial infarction myocardial cells apoptosis. We also confirmed nourishing qi traditional Chinese medicine could enhance stem cells to mend injured heart. Although the relationship between Sirt1 and cardiac stem cells in myocardial infarction has not been reported at home and abroad yet. So this study will use Sirt1 knockout and transgenic mice to model myocardial infarction, which are treated in removing blood stasis for regeneration traditional Chinese medicine. We will utilize molecular biology technology, flow cytometry technology, etc, analyze apoptosis pathway and oxidative stress, etc, and observe the proliferation change of cardiac c-kit-positive/Lin-negative stem cells. The objectives are to reveal the relationship of traditional Chinese medicine treatment effect, Sirt1 expression and cardiac c-kit-positive/Lin-negative stem cells change, and elucidate the mechanism of treating myocardial infarction in removing blood stasis for regeneration traditional Chinese medicine. The study will confirm our proposed hypothesis that myocardial infarction could induce cardiac stem cells proliferation, while increased expression of Sirt1 could enhance the regenerative effect of cardiac stem cells, and the traditional Chinese medicine treatment in removing blood stasis for regeneration could target at cardiac Sirt1-stem cells，all of which together could realize the effect of mending ischemic and infarcted myocardium. This study will provide a new idea to prevent and treat myocardial infarction.

影响心肌梗死（MI）预后的主要病理因素是受损心肌功能丧失，如何促进心肌细胞再生是影响预后的关键。已知Sirt1能在应激条件下减少细胞凋亡，而心脏干细胞有研究证实也可分化为良好的心肌。中药在MI治疗中有着重要地位，课题组通过前期国家自然基金探索资助项目发现祛瘀生新法能调控Sirt1抑制细胞凋亡，促进干细胞修复受损心肌。但Sirt1与心脏干细胞在MI中的相关性尚无报道。本课题拟用Sirt1基因敲除与转基因小鼠复制MI模型，通过祛瘀生新中药干预，采用分子生物学、流式细胞术等方法，以细胞凋亡通路、氧化应激等为切入点，观察心脏干细胞增殖变化，揭示预后与Sirt1表达、心脏干细胞变化的关系，将证实"MI能诱使CSC增殖，Sirt1表达增多可提高心脏干细胞再生能力，祛瘀生新法具有靶向Sirt1-心脏干细胞效应，从而实现共同修复缺血坏死心肌的作用"假说，为中医药防治MI及其作用机制研究提供新思路。

研究背景：心肌梗死（MI）发病率和死亡率在我国呈明显上升趋势，是引起死亡主要疾病之一，严重危害百姓的健康。影响心肌梗死（MI）预后的主要病理因素是受损心肌功能丧失，因此如何减少缺血心肌细胞损伤，促进受损心肌细胞修复是影响预后的关键。已知Sirt1 能在应激条件下减少细胞凋亡，而心脏干细胞有研究证实也可分化为良好的心肌。本课题从SIRT1与心脏干细胞的角度探讨血府逐瘀汤胶囊治疗急性心肌梗死的机制，为中医药防治MI及其作用机制研究提供新思路。. 主要研究内容：分别采用正常、SIRT1基因敲除与转基因三种不同小鼠，复制MI模型，通过祛瘀生新中药干预，采用分子生物学、流式细胞术等方法，以细胞凋亡通路、氧化应激等为切入点，评价祛瘀生新法治疗对于心肌梗死模型小鼠的疗效，并进一步揭示预后与Sirt1 表达、心脏干细胞变化的关系。. 重要研究结果、关键数据及其科学意义： . （1）使用小动物活体成像测量心肌缺血面积，结果显示，与模型组相比，假手术组、血府逐瘀组的心肌缺血面积明显减少（P<0.01），说明血府逐瘀胶囊可以减少心肌梗死模型小鼠的心肌缺血面积，具有较好的治疗效果。. （2）使用心脏超声评估心脏功能，结果显示，与模型组相比，假手术组、血府逐瘀组的左室短轴率（FS）、左室射血分数（EF）显著增高（P<0.01），说明血府逐瘀胶囊可以提高心肌梗死模型小鼠的心脏功能，改善其预后。. （3）本研究TUNEL检测结果显示，血府逐瘀胶囊可以抑制缺血心肌细胞的凋亡。活性氧检测结果显示，血府逐瘀胶囊可以减少因缺血缺氧导致氧化应激活性氧的产生。免疫组化、RT-PCR、Western-blot结果显示，能够激活SIRT1信号通路，促进信号通路MnSOD2、bcl-xl抑凋亡蛋白表达，同时抑制FOX01、P53、Bax等促凋亡蛋白表达，说明血府逐瘀胶囊可能通过作用于SIRT1信号通路，发挥其治疗急性心肌梗死的目的。. 本项研究在中医祛瘀生新思想指导下治疗心肌梗死，为中医药干预心肌梗死作用机制研究及临床应用提供了科学依据。