NR4A1/FUNDC1/线粒体自噬通路在心肌缺血再灌注损伤中的作用及机制研究

Numerous evidences have highlighted that the core pathogenesis of myocardial reperfusion injury is associated with the imbalance of mitochondrial homeostasis that is mainly regulated by mitophagy and mitochondrial fission. Our previous studies confirmed that FUNDC1/mitophagy pathway has a protective effect on myocardial reperfusion injury, but its upstream regulatory mechanisms and downstream molecular events remain unclear. It has been previously demonstrated that NR4A1, a novel mitochondrial homeostasis regulator, can inhibit mitophagy in a variety of disease models, but whether it can regulate mitophagy via FUNDC1 and promote myocardial reperfusion injury has not been reported. In downstream events, it is unclear that whether FUNDC1/mitophagy can protect myocardium from the reperfusion injury by inhibiting mitochondrial fission. Thus, we knockdown and overexpress target proteins in vivo and in vitro to confirm that NR4A1 can inhibit FUNDC1/mitophagy pathway, promote mitochondrial fission, impair mitochondrial homeostasis and ultimately aggravate myocardial reperfusion injury. This subject will provide potential new molecular targets for the prevention and treatment of myocardial reperfusion injury, and further enrich the mechanisms of myocardial reperfusion injury that are related to mitochondrial homeostasis.

线粒体稳态失衡是心肌再灌注损伤的核心病理机制，线粒体自噬和线粒体分裂在线粒体稳态调控中起着关键作用。本团队前期研究证实FUNDC1/线粒体自噬通路对心肌再灌注损伤具有保护作用，但其上游调控机制和下游分子事件仍不明确。NR4A1是新型的线粒体稳态调控因子，本团队前期在多种疾病模型下都已证实NR4A1可以抑制线粒体自噬，但其能否通过FUNDC1调节线粒体自噬、进而促进心肌再灌注损伤未见报道。下游事件中，FUNDC1/线粒体自噬能否通过拮抗线粒体分裂，进而保护再灌注心肌还不明确。本课题利用靶蛋白敲低和过表达等手段，分别在动物、细胞、分子水平，阐明NR4A1通过抑制FUNDC1/线粒体自噬通路，促进线粒体分裂，最终诱发线粒体稳态失衡、加重心肌再灌注损伤的分子机制。本课题将为心肌再灌注损伤提供潜在新型分子干预靶点，进一步丰富基于线粒体稳态失衡为中心的心肌再灌注损伤信号机制。

急性心梗会破坏包括心肌、心脏微循环在内的组织，造成严重的公共卫生负担。在心肌细胞转归还的调控中，线粒体起着不可替代的作用。线粒体通过自身的损伤性分裂、保护性自噬清除以及改善性融合整合，对再灌注损伤信号进行修饰，参与心脏组织的病理生理进程，介导心肌和内皮的凋亡或存活，从而精确调节心肌和微循环的结构和功能。本研究利用C57小鼠、WT大鼠、NR4A1和OPA1低表达小鼠, 建立心肌再灌注损伤模型。同时利用小鼠心房肌细胞系（HL-1），在体外建立缺氧复氧损伤模型。利用免疫组化、免疫荧光、western blot、qPCR、干扰RNA敲低、超微结构电镜等实验方法，观察线粒体能量代谢、线粒体ROS生成、线粒体cyt-c渗出、线粒体膜电位、mPTP开放、线粒体分裂、线粒体融合、炎症反应通路等变化。研究结果证实NR4A1在心肌缺血再灌注损伤后显著上调，抑制NR4A1表达可恢复减少心肌细胞凋亡、改善线粒体功能与形态、减小梗死面积、降低氧化应激水平和炎症反应。机制上， NR4A1可以下调线粒体融合相关蛋白Opa1的表达情况，进而抑制线粒体融合，促进线粒体碎片化，加剧线粒体分裂，导致线粒体内容物的释放，损伤线粒体功能与结构，造成更严重的缺血再灌注损伤。本研究确立了基于NR4A1调控的线粒体稳态为核心的微循环再灌注损伤病理生理机制，为临床干预心肌微循环再灌注损伤提供了潜在的分子靶点。