基于线粒体自噬调控的苗药都木油活性成分抗MIRI的分子机制研究

Pharmacological preconditioning (PPC) is the effective strategies against myocardial ischemia/reperfusion injury (MIRI)。Numerous studies have shown that the myocardial ischemia tolerance of PPC was involved mitophagy which regulated cooperatively by PINK1-Parkin pathway and the mitophagic receptors Bnip3. In our previous national natural science project, we not only found the anti-MIRI effects by orientin, the active ingredient of Ndutmongxyoux DC., may due to anti-apoptosis and autophagy, also found that mitophagy was the major form of autophagy in myocardial cel l. This project takes the effects of mitophagy on the change of mitochondrial quality as a breaking-through point, takes the interactive regulation of autophagy and apoptosis mediated by PINK1-Parkin pathway and the mitophagic receptors Nix and Bnip3 as main research path, using some advanced technologies, such as transmission electron microscope, fluorescence microscope, confocal laser scanning microscope and Western blot, and in combination with system science research method，to investigate the effects of mitophagy induced by Ndutmongxyoux DC., to explore the network mechanism of interactive regulation of autophagy and apoptosis induced by Ndutmongxyoux DC. at the level of subcellular structure , and to further test and verify the hypothesis "the core mechanism of anti-MIRI by Ndutmongxyoux DC. are the moderate induction of mitophagy and inhibition of apoptosis, to improve mitochondrial quality, finally enhance the ischemia/reperfusion tolerance". This study would provide the pharmacological evidence for Ndutmongxyoux DC.against MIRI.

药物预处理（PPC）是心肌缺血再灌注损伤（MIRI）的有效防治措施。众多研究表明，PPC增强心肌缺血耐受与PINK1-Parkin信号通路及相关通路Bnip3协同调控线粒体自噬有关。在上一个国家自然基金项目研究中，我们不仅发现苗药都木油活性成分荭草苷抗MIRI作用与增强自噬及抗凋亡相关，还发现线粒体自噬是心肌细胞主要自噬方式。在前期工作基础上，本项目以线粒体自噬对线粒体质量变化的影响为研究切入点，以PINK1-Parkin信号通路和线粒体自噬受体Bnip3共同参与的自噬与凋亡调控网络为主要研究路径，采用基因沉默、透射电镜、激光共聚焦、Western Blot等技术，结合系统科学研究方法，从亚细胞结构探寻都木油对自噬/凋亡互调节的网络调控机制，并验证"苗药都木油抗MIRI的核心是适度诱导线粒体自噬，抑制细胞凋亡，改善线粒体质量，增强MIRI耐受性"的假说，为苗药都木油防治MIRI提供实验依据

研究表明，心肌缺血再灌注（myocardial ischemia reperfusion injury，MIRI）诱导的过度的自噬不仅不能保护心肌细胞甚至会造成更严重的心肌损伤。本课题组前期研究发现药都木油活性成分荭草苷对心肌缺血再灌注损伤有保护作用，且作用机制均与调控细胞凋亡和细胞自噬有关。本项目通过建立在体心肌缺血再灌注损伤模型，采用电镜、荧光显微镜、酶标仪、TNUEL、Western blot、免疫共沉淀等技术方法，研究MIRI过程中线粒体质量、细胞凋亡和细胞自噬的变化情况，并深入研究了荭草苷对线粒体自噬相关的PINK1-Parkin和Bnip3通路的调控机制。实验结果表明：① 荭草苷预处理能明显提高线粒体膜电位，减小mPTP开放程度，升高线粒体呼吸链复合物含量，减轻心肌线粒体损伤，明显改善线粒体质量；② 荭草苷通过下调促凋亡蛋白Bax和Bak并上调抑凋亡蛋白Bcl-2和Bcl-xl来抑制心肌细胞凋亡，同时还能抑制MIRI心肌细胞过度自噬；③ 荭草苷通过抑制Parkin和p62的表达，减弱Parkin和p62的结合程度，来抑制PINK1-Parkin信号通路调控的心肌细胞线粒体自噬；④ 荭草苷通过降低Bnip3的表达，来抑制Bnip3信号通路调控的心肌细胞线粒体自噬。综上所述：荭草苷可能是通过调控线粒体凋亡和线粒体自噬，改善了线粒体质量，从而发挥抗心肌缺血再灌注损伤的作用，验证了“苗药都木油活性成分荭草苷防治MIRI的核心是适度诱导线粒体自噬，抑制细胞凋亡，改善线粒体质量，增强MIRI耐受性”的假说，为苗药都木油防治MIRI提供实验依据。