碘化N-正丁基氟哌啶醇通过JNK调节线粒体ROS自持环路拮抗缺血再灌注心肌线粒体氧化应激

Oxidative stress caused by ROS is an important pathological mechanism of myocardial ischemia/reperfusion (I/R) injury. Our previous studies showed that F2, an innovative compound, antagonizes myocardial I/R injury by regulating ROS/JNK/Egr-1 pathway from cytoplasm to nuclei. It was reported recently that JNK in cytoplasm promotes liver injury by activating Sab/PTPs/SHP1/ROS signaling pathway in mitochondrion. ROS in myocardium following I/R comes mainly from mitochondrion, which are prime criminals and also victims for myocardial I/R injury, nevertheless, the mechanisms for sustained ROS production in mitochondrion remains unclear. Based on literature and our results, we hypothesized that an ROS/JNK/Sab/PTPs/Src/Respiratory Chain Complexs/ROS signaling pathway is activated in myocardial mitochondrion following I/R, which results in oxidative stress in mitochondrion and then I/R injury; F2 antagonizes mitochodrial oxidative stress, and subsequently maintains calcium homeostasis and protects myocardium from other I/R associated injuries by regulating JNK-mediated self-sustained ROS production loop in mitochondrion. Therefore, the purpose of this study is to elucidate the molecular mechanism for sustained ROS production and its pathogenicity after myocardial I/R on myocardial I/R model in vivo and in vitro, and the effects of F2 on them. The study will help to better understand the molecular mechanism of myocardial I/R, and meanwhile provide the theory basis for F2 to apply other oxidative stress-associated disorders.

活性氧（ROS）引起的氧化应激是心肌缺血再灌注（I/R）损伤重要病理机制。我们发现创新化合物F2可抑制I/R所致胞浆至胞核ROS/JNK/Egr-1通路，保护心肌。国外新近发现胞浆JNK可通过线粒体内Sab/SHP1/Src/ROS通路致肝损伤。心肌线粒体是ROS最主要来源、I/R损伤的元凶，也是氧化应激受累者，但线粒体ROS持续生成的具体机制不明。我们提出假说：I/R心肌线粒体存在ROS/JNK/Sab/PTPs/Src/线粒体复合体/ROS通路，导致线粒体自身氧化应激，启动心肌I/R损伤；F2通过JNK抑制以上ROS促ROS自持环路，拮抗线粒体氧化应激，由此维持钙稳态，拮抗心肌I/R其它损伤。本项目拟在整体和体外心肌I/R模型上，探讨心肌I/R时线粒体ROS持续生成分子机制及致病性，并研究F2于其中的作用，这将深入揭示心肌I/R损伤分子机制，为F2用于其它氧化应激相关疾病提供理论基础。

活性氧（ROS）积聚引起的氧化应激是心肌缺血再灌注（I/R）损伤的重要病理生理过程。有研究表明，胞浆中JNK可结合到线粒体外膜Sab蛋白上，导致线粒体内Src失活，进而抑制电子传递链，使线粒体ROS生成增加，引起肝脏损伤。我们前期研究结果表明，心肌来源的H9c2细胞及心脏微血管内皮细胞缺氧/复氧（H/R）时，可引起胞浆中ROS生成及p-JNK表达增加。同时发现，我们的专利化合物F2可以拮抗大鼠整体的I/R损伤及离体心肌细胞H/R损伤，其机制包括降低胞浆ROS水平，抑制JNK活化，但涉及的具体分子机制不清楚。本研究发现心肌I/R时胞浆ROS含量增加可引起JNK的磷酸化激活及线粒体转位，促使线粒体相关的JNK/Sab/Src/ROS信号通路活化，导致线粒体氧化应激损伤及最终的心肌组织I/R损伤。F2可以通过抑制再灌注所致的胞浆ROS/JNK/Sab/Src/线粒体ROS信号环路，拮抗心肌再灌注诱导的线粒体氧化应激损伤，进而保护I/R心肌；同时研究发现，具有降低线粒体内ROS水平的线粒体靶向抗氧化剂MT可以抑制I/R所致的胞浆JNK激活与线粒体转位，保护线粒体结构与功能，抑制心肌肌纤维溶解，抑制炎症细胞渗出，改善线粒体超微结构的损害，缩小心肌梗死面积，提高左心室的射血分数和短轴缩短率，验证了线粒体产生的ROS可通过引起胞浆JNK的磷酸化激活及线粒体转位，引起心肌I/R损伤；研究还发现F2可以通过抑制人源的脐静脉内皮细胞（HUVECs）中ROS/JNK/NF-κB信号通路，保护其线粒体的结构与功能，维持线粒体融合与分裂的动态平衡，拮抗细胞的H/R损伤，改善血管内皮细胞的功能。本项目不仅揭示了心肌I/R损伤新的分子机制，丰富了F2抗心肌I/R损伤的作用机制，使其更具开发价值，也为其它内皮功能障碍相关疾病发病机制的阐明提供了启示，同时有助于拓展F2在其它相关疾病治疗中的应用。