缺氧细胞中mTORC1通过下调EF-Tumt表达引起线粒体损害的分子机制研究

Ischemia/hypoxia injury is common in severe burn, and mitochondrial damage plays an important role in mediating ischemic/hypoxic myocardial injury. However, the underlying mechanisms remain to be illustrated. Our previous studies have found that hypoxia causes decreased expression of EF-Tumt in cardiomyocytes, and down-regulation of EF-Tumt level by siRNA leads to mitochondrial damage under normoxia condition, suggesting that decreased expression of EF-Tumt leads to mitochondrial damage in hypoxic cardiomyocytes. It has been reported that hypoxia decreased mTOR activities in multiple cells, to explore the key molecules at the up-stream of EF-Tumt, we treated cardiomyocytes with a mTOR complex 1 (mTORC1) inhibitor-rapamycin, and found that rapamycin decreased EF-Tumt expression in cardiomyocytes. Thus, we hypothesize that mTORC1 plays an important role in mediating mitochondrial damage via down-regulation of EF-Tumt level in hypoxic cardiomyocytes. To confirm this hypothesis, firstly, we investigated the changes of EF-Tumt level and mitochondrial functions regulated by mTORC1; Secondly, we down or up-regulate the mTORC1 activity through constructing Raptor or TSC2 RNAi vector in H9C2 cell, and investigate its role in regulating EF-Tumt expression and mitochondrial functions under hypoxia; Further, we investigate the pathways by which decreased mTORC1 activity causes mitochondrial damage under hypoxia. Our study will provide a new pathway in prevention of mitochondrial damage under hypoxia .

烧伤早期损害的实质是缺血缺氧损害，线粒体受损是核心步骤，其机制与调控环节尚不明确。本课题组前期发现，缺氧使线粒体翻译延伸因子Tu（EF-Tumt）表达降低；常氧时，低表达EF-Tumt即引起线粒体损害，提示EF-Tumt低表达介导缺氧后线粒体损害。文献报告，缺氧使雷帕霉素靶蛋白（mTOR）活性降低，本组进一步发现单用mTOR复合体1（mTORC1）抑制剂雷帕霉素即可引起EF-Tumt表达下降。基于上述事实本课题提出"缺氧通过mTORC1调控EF-Tumt表达引起线粒体损害"的假设。拟明确mTORC1对EF-Tumt表达与线粒体功能的调控，构建mTORC1稳定活化/失活细胞系，探讨缺氧时mTORC1活性对EF-Tumt表达与线粒体功能的影响；在此基础上，进一步干预EF-Tumt表达，阐明mTORC1调控线粒体功能的分子机制。通过本研究，有望从新的角度揭示缺氧后线粒体损伤发生机制与调控环节。

缺血缺氧是介导烧伤早期心肌损害的重要因素，其作用机制未明。我们发现，在体大鼠模型中，严重烧伤引起心肌组织EF-Tumt表达降低。为明确其在心肌细胞中的作用，我们体外培养大鼠乳鼠原代心肌细胞，以小干扰RNA法下调EF-Tumt表达发现，EF-Tumt表达下调通过影响呼吸链复合体I亚单位蛋白表达与酶活性、增加细胞中ROS产生与蛋白羰基化水平导致心肌细胞损伤。进一步发现，缺氧早期可引起心肌细胞中EF-Tumt表达下调，以腺病毒上调EF-Tumt表达可通过减少缺氧心肌细胞中线粒体ROS的产生而发挥保护作用。此外，缺氧可引起心肌细胞中mTORC1信号失活、AMPK信号激活，并伴随自噬增多与自噬流受损并存的现象。在此基础上，我们探讨了缺氧心肌细胞中自噬流受损发生的分子机制，发现缺氧使心肌细胞中ATP与NAD水平均降低。常氧下，分别以药物干预降低细胞内ATP与NAD水平后发现，ATP耗竭可使自噬减少，而NAD耗竭则引起心肌细胞自噬流受损。并且，向缺氧心肌细胞中分别补充ATP与NAD发现，补充ATP不能有效改善自噬流受损，而补充NAD则可有效改善自噬流受损。提示缺氧后心肌细胞自噬流受损可能与细胞内NAD水平降低相关。这可能是调控心肌细胞缺氧损伤的新靶点。

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