甘青铁线莲中活性成分APG通过PKCε/mKATP通路减轻心肌缺血/再灌注损伤的机制研究

Myocardial Ischemia-reperfusion injury (MI/RI) is one of the urgent problems to be solved. Administration of pharmacological agents mimicking the effect of ischemic preconditioning, which was defined as pharmacology preconditioning (PPC), may provide an effective means to rescue the injured myocardial insulted by I/R challenge. We put forward a view that the key role of ‘QI’ and ‘LONG’ in treating ‘XiongBi’ syndromes in traditional Chinese medicine and Tibetan medicine theory may have some relationship with the crucial mechanism of protecting mitochondrial functions in PPC. It has provided us with innovated ideas to study the heart protection mechanisms of traditional medicine. Recently, a flavonoid glycoside named APG was isolated from the Tibet herbal medicine C tangutica in our lab. We found for the first time that APG has potent myocardial protection effect against MI/RI in rats. Biochemical analysis result showed that APG decreased the MDA and increased the SOD levels; Bcl-2/Bax ratio and activity of Caspase-3 were raised after APG treatment in MI/RI. Pre-Experiment showed that APG improved the expression of PKCε in mitochondrial and maintained the mitochondrial membrane potential. Whether APG had PPC effect and protected myocardial by activating PKCε expression and then affected the mKATP pathway to preserve the function of mitochondria, was needed further studies. This study planed to apply the Transmission Electron Microscopes, Laser Scanning Confocal Microscopy, Western Blotting and protein electrophoresis method and technic to explore the potential role of PKCε/mKATP pathway in myocadiol protection of APG on the animal, cellular and molecular levels. Our study will provide theoretical basis for utilization APG in clinical in the future.

心肌缺血再灌注损伤（MI/RI）是临床亟待解决的问题。药理性预适应(PPC)效应中保护线粒体作用与中医“气”、藏医“隆”在治疗胸痹症中的关键作用理论有一定联系，这为我们进行传统药心肌保护机制研究提供了思路。本课题组从藏药甘青铁线莲中分离出一种黄酮苷类化合物APG。通过大鼠MI/RI模型首次发现APG具有较强心肌保护作用，研究显示APG能降低MDA以及提高SOD水平，升高Bcl-2/Bax比值，降低Caspase-3活性。预实验结果表明APG能提高线粒体中PKCε表达，维持线粒体膜电位。APG是否具有PPC，通过激活PKCε作用于mKATP通路，保护线粒体发挥心肌保护作用，有待验证。本项目拟采用透视电子显微镜、激光共聚焦显微镜扫描、Western blot、蛋白电泳等手段，在整体、细胞、分子水平研究APG是否通过PKCε/mKATP通路产生心脏保护效应，本研究将为APG应用于临床奠定基础。

心肌缺血再灌注损伤（MI/RI）是临床亟待解决的问题。药理性预适应(PPC)效应中保护线粒体作用与中医“气”、藏医“隆”在治疗胸痹症中的关键作用理论有一定联系，这为我们进行传统药心肌保护机制研究提供了思路。本课题组从藏药甘青铁线莲中分离出一种具有心肌保护活性黄酮苷类化合物APG。通过细胞和动物模型系统地研究其心肌保护机制。在MI/RI细胞模型中，APG于造模前24h给予预处理，结果显示其能够显著提高原代细胞活性及减轻细胞凋亡。采用western blot检测蛋白表达，APG能促进Bcl2表达，抑制Bax和Caspase3表达；促进线粒体内SDH, COX生成，增加线粒体SOD活性，抑制H2O2形成和降低MDA含量；APG能够促进PKCε磷酸化及线粒体转位，促进其下游通路Nrf2核转位、HO-1表达上调。通过PKCε靶向siRNA转染方法阻断，上述效应均被抑制。在动物水平，采用C57BL/6小鼠MI/R损伤模型，造模前腹腔给予APG预处理，结果显示APG显著改善MIRI造成的心功能降低趋势，增加LVEF和FS心功能指标，能够促进PKCε磷酸化及线粒体转位、Nrf2核转位，HO-1表达上调，促进心肌组织线粒体内SDH、COX、SOD蛋白表达，降低H2O2和MDA表达。通过给予PKCε抑制剂ε-V1-2处理后，以上效应均被抑制。基于以上研究结果，结合文献回顾及课题假说，我们提出结论：APG是一种具有药理性预适应活性的天然产物，其发挥心肌保护作用机制包括一方面通过激活PKCε和促进其向线粒体转位，从而减轻线粒体氧化应激损伤；另一方面通过激活PKCε进而激活Nrf2向核转位，进一步上调HO-1从而发挥抗凋亡保护作用，两种保护机制协同发挥了较强的心肌保护作用。该结果对于阐明甘青铁线莲作为藏药材治疗心脏病提供了一定的理论依据，同时对于APG的进一步开发和临床转化提供了理论基础。