活血解毒中药配伍调控心肌细胞自噬改善冠脉无复流的作用机制研究

Coronary No-reflow (CNR) is one of the major reasons for the failure in the clinical treatment of coronary heart disease by Perrutaneous Coronary Intervention (PCI). CNR-induced the robust autophagy of cardiomyocytes can lead to cell death, which is strongly related to the inactivation of PI3K/Akt/mTORC1 signaling pathway. Our previous studies found that Chinese herbal compound for detoxifying and activating blood circulation treatment could significantly prevent CNR phenomenon resulting from ischemic reperfusion (I/R), suggesting that Chinese herbal compound for detoxifying and activating blood circulation may exert its protection effects via modulating the PI3K/Akt/mTORC1 signaling pathway. In this present project we will evaluate the in vivo and in vitro protection effects of Chinese herbal compound for detoxifying and activating blood circulation on myocardial injury resulting from I/R. In addition, we will determine the effect of Chinese herbal compound for detoxifying and activating blood circulation on the PI3K/Akt/mTORC1 signaling pathway as well as the expression of autophagy-related target genes. Our findings in this study will provide new insight into the molecular mechanism of Chinese herbal compound for detoxifying and activating blood circulation of protection effecte on cardiomyocytes jury following I/R. It will provide further experimental evidence for clinically treatment of CNR-induced by ischemic reperfusion.

冠状动脉无复流（CNR）可引起心肌细胞发生过度自噬进而导致心肌细胞死亡，是冠心病介入治疗失败的主要原因，与PI3K/Akt/mTORC1通路失活所引起的细胞过度自噬密切相关。课题组前期研究发现活血解毒中药川芎、赤芍和黄连的配伍能够有效防治缺血再灌注损伤中的CNR现象，据此提出假说：调控PI3K/Akt/mTORC1通路的活化、抑制心肌细胞的自噬是活血解毒中药防治CNR的重要机制。本课题拟分别采用心肌缺血再灌注和缺氧-复氧培养构建无复流动物和细胞模型，通过检测心肌无复流/梗死面积和细胞活力等方法评估活血解毒中药配伍对心肌细胞体内外的保护作用；通过电镜、免疫荧光染色、Q-PCR和Western Blot等技术研究活血解毒中药配伍对心肌细胞自噬的影响以及对PI3K/Akt/mTORC1信号通路的调控作用。研究结果将有助于阐明活血解毒中药配伍防治CNR的作用机制，为临床应用提供实验依据。

冠状动脉无复流（CNR）可引起心肌细胞过度自噬，是冠心病介入治疗失败的重要原因。本项目以PI3K/Akt/mTORC1信号通路为切入点，通过构建无复流动物和细胞模型，采用电镜、免疫荧光染色、Q-PCR和Western Blot等技术，深入研究活血解毒中药配伍抑制心肌细胞自噬改善冠脉无复流的作用和机制。体内实验采用I/R手术制备大鼠CNR模型，生化检测显示大鼠血清CK-MB和cTnT显著升高，活血解毒中药灌胃后血清CK-MB和cTnT显著降低。硫磺素S和硝基四氮唑蓝染色结果显示活血解毒中药可以显著减少大鼠心肌梗死面积和无复流区域面积。Western Blot结果显示活血解毒中药可以显著降低CNR大鼠心肌自噬相关蛋白Beclin-1、LC3II/I和凋亡相关蛋白Cleaved Caspase3、β-catenin、p-p65的表达，升高p62、p-AKT、p-mTOR、Bcl-2的表达。PCR检测发现活血解毒中药能够显著降低大鼠心肌组织Beclin-1和LC3 mRNA的表达，增加Bcl-2 mRNA的表达，降低CNR大鼠心肌细胞自噬和凋亡水平。体外实验采用缺氧复氧刺激H9C2细胞，并给予活血解毒中药预处理，电镜观察显示细胞自噬体减少，线粒体分解现象缓解；流式细胞术检测发现细胞的凋亡水平降低。细胞生长活性检测和荧光显微镜观察发现中药和PI3K抑制剂联合预处理，对心肌细胞的自噬和凋亡的抑制效果会减弱，中药和溶酶体抑制剂联合预处理，对心肌细胞的自噬和凋亡的抑制效果会增强。Western Blot结果显示，经过缺氧复氧处理后，细胞自噬相关蛋白Beclin-1、LC3II/I表达增加，p62、p-AKT、p-mTOR表达降低；凋亡相关蛋白Cleaved Caspase3、β-catenin、p-p65表达增加，Bcl-2表达降低。但经过中药预处理后，自噬和凋亡水平均显著降低。PCR结果显示，H9C2细胞经过缺氧复氧处理后，Beclin-1和LC3 mRNA表达显著增加，Bcl-2 mRNA表达显著降低，经过中药预处理后，细胞自噬和凋亡水平均显著降低。由此证实，活血解毒中药配伍通过调控PI3K/Akt/mTOR 信号通路的活化，抑制心肌细胞的自噬和凋亡来发挥其改善冠脉无复流的作用。研究结果将为活血解毒中药的临床应用提供实验依据。