基于SIRT1信号通路探讨活血温通方联合MSCs移植改善急性心梗后梗死区微环境的作用机制

A large number of cardiomyocyte cell deceases severely constrain the recovery of cardiac function after acute myocardial infarction (MI). The loss of cardiomyocyte cell is not sufficient by supplementation of stem cell in situ. Although stem cell transplantation is promising currently, the low survival rate of mesenchymal stem cells (MSCs) that after transplantation into the heart seriously affects the therapeutic effect due to the poor microenvironment of the infarction area. It has been found that the combination of blood-activating traditional Chinese medicine with MSCs transplantation has positive significance in the treatment of MI. Our team found that the traditional Chinese medicine of activating blood and yang has a better curative effect than the pure traditional Chinese medicine that promotes blood circulation and removing blood stasis in the acute phase of MI on inheritance of prestigious TCM doctors’ experiences. Therefore, based on the basic pathogenesis of Insufficiency of Yang and Endogenous Excess of Yin Pathogens in thoracic obstruction, and basic studies in Earlier Stage on Eliminating Blood Stasis to Promote Regeneration of Blood promote cardiomyocyte regeneration by Improving Sirt1 Signaling Pathway, the MI model was used to observe changes in Sirt1 signaling pathway closely related to cell regeneration from both in vivo and in vitro pathways according to the application for the combination of formula of activating blood and yang with MSCs transplantation by empirical formula of famous veteran doctors of TCM. So we could to understand the effect of this prescription on survival, paracrine function, and cardiac function of MSCs after transplantation. This will confirm the hypothesis that formula of activating blood and yang can improve the microenvironment of ischemia and hypoxia after MI, promoting the survival and paracrine function after stem cell transplantation, and then has synergistic with effect recovery of heart function. It provides new ideas for the prevention and treatment of myocardial infarction.

大量心肌细胞死亡严重制约了急性心梗（MI）后心功能恢复，这种心肌细胞丢失仅依靠原位干细胞的补充是不够的。目前干细胞移植尽管很有前景，但由于梗死区恶劣的微环境，导致间充质干细胞（MSCs）移植到心脏后存活率低，严重影响疗效。既往发现活血类中药联合MSCs移植在MI治疗中有积极意义。本团队在名老中医经验传承中发现活血温通中药较单纯活血化瘀类中药在心梗急性期疗效更好。由此，拟基于胸痹“阳微阴弦”基本病机，在前期研究“祛瘀生新法通过改善Sirt1信号通路促进心肌细胞再生”基础上，运用名中医经验方活血温通方联合MSCs移植作用于心梗模型，从体内和体外途径观察与细胞再生密切相关的Sirt1信号通路变化，以了解该方对MSCs移植后存活、旁分泌功能以及心功能的影响。这将证实“活血温通方能够改善心梗后局部缺血缺氧微环境，促进干细胞移植后的存活和旁分泌功能，从而协同恢复心功能”假说，为防治心梗提供新思路。

研究背景：干细胞移植治疗心梗（MI），有望修复坏死的心肌组织，改善心功能，但由于梗死区恶劣的微环境，导致间充质干细胞（MSCs）移植到心脏后存活率低，影响疗效。既往发现中药联合MSCs移植在MI治疗中有积极意义。由此，运用名中医经验方活血温通方联合MSCs移植作用于心梗模型，从体内和体外途径观察该方对MSCs移植后存活、旁分泌功能以及心功能的影响。.主要研究内容：通过体内和体外实验，基于Sirt1 信号通路，采用活血温通中药联合MSCs移植治疗MI，明确活血温通方对MSCs移植后存活和旁分泌功能的影响，Sirt1信号通路表达变化，以及对心功能的恢复作用。.重要结果、关键数据及其科学意义：.（1）使用全扫显微镜观察，活血温通方联合MSCs移植组比MSCs移植组镜下红色荧光分布更广，红色荧光与蓝色荧光重叠的数量明显更多，说明活血温通方可以提高MSCs移植后的存活率。使用心脏超声评估心功能，结果显示，活血温通方联合MSCs移植组EF和FS比BMSCs移植组和活血温通方组改善明显，说明活血温通方联合MSCs移植可协同改善MI大鼠的心功能。.（2）TUNEL染色、免疫组化、天狼猩红染色显示，活血温通方联合MSCs移植可协同减轻MI大鼠心肌细胞凋亡，促进MSCs移植后梗死边缘区α-SMA和CD31的表达以促进血管新生，以及协同对抗心肌纤维化的作用。ELISA检测血清生长因子显示，活血温通方联合MSCs移植可提高VEGF和bFGF水平，活血温通方能促进MSCs移植后的旁分泌功能。Western blot检测心肌组织Sirt1信号通路蛋白表达，活血温通方联合MSCs移植组Sirt1的表达增多，NF-kB、Foxo1和p53的表达降低。.（3）ROS检测，显示活血温通方处理组氧化应激水平降低。凋亡和线粒体膜电位检测，红色荧光与绿色荧光显示其细胞存活状态，活血温通方处理组，细胞凋亡水平改善。ELISA检测细胞上清因子显示，活血温通方可提高心肌细胞的bFGF水平，活血温通方能促进心肌细胞的分泌功能。Western blot检测心肌细胞Sirt1信号通路蛋白表达，活血温通方组Sirt1的表达增多，NF-kB、Foxo1和p53的表达降低，线粒体生物发生相关蛋白PGC-1a增多，VDAC1表达降低。.本研究阐明了活血温通中医治则治法的科学内涵，为中医药联合干细胞移植治疗MI提供科学依据。