探讨活性氮介导的过度线粒体自噬在脑缺血再灌注损伤中的作用及抗氧化剂柚皮苷的干预效应

Mitophagy, a selective autophagy of mitochondria, is the new direction of stroke research. Cerebral ischemic-reperfusion injury produces large amount of free radicals and mediates mitophagy. Mitophagy is considered as a survival attempt to remove damaged mitochondria to keep cell homeostasis. Intriguingly, excessive mitophagy initiates cell death. Reactive nitrogen species (RNS) are important components of free radicals and play critical roles in mitochondrial damages during cerebral ischemia-reperfusion injury. However, the roles of RNS in mediating mitophagy and its impact on cerebral ischemia-reperfusion injury are unknown yet. Our preliminary data revealed that the levels of RNS and mitophagy were significantly increased during cerebral ischemia-reperfusion injury, and treatment of FeTMPyP, a representative peroxynitrite decomposition catalyst (PDC), inhibited excessive mitophagy and alleviated neuron damages. Furthermore, antioxidant naringin revealed to scavenge RNS, attenuate mitophagy and protect ischemia-reperfused brains. Therefore, we hypothesize that RNS could be a player in mediating excessive mitophagy and aggravating cerebral ischemia-reperfusion injury, and antioxidant narignin could inhibit RNS-mediated excessive mitophagy and protect against cerebral ischemia-reperfusion injury. We will test the hypothesis with following three strategies: (1) To illustrate the role of excessive mitophagy in cerebral ischemia-reperfusion injury by intervening mitophagy level using drugs and ATG5 siRNA technique; (2) to explore the effects of RNS on mediating excessive mitophagy and the regulations of PINK1/Parkin-mediated mitophagy signaling pathway by using RNS donor, peroxynitrite decomposition catalyst and RNS scavenger; (3) to identify the therapeutic strategy of targeting RNS-mediated excessive mitophagy as a novel direction of treatment against cerebral ischemia-reperfusion injury by using naringin as an example.

线粒体自噬是脑卒中研究新方向，脑缺血再灌注产生大量自由基诱导线粒体自噬。通过自噬清除受损线粒体维持细胞稳态，而过度线粒体自噬则会导致细胞死亡。活性氮是重要的自由基，在线粒体损伤过程中起关键作用，目前有关活性氮调控脑缺血再灌注损伤中线粒体自噬的研究仍是空白。前期研究表明：脑缺血再灌注明显提高活性氮水平及线粒体自噬程度，且过氧亚硝基解构剂FeTMPyP干预后能抑制线粒体自噬，减轻神经元损伤；同时抗氧化剂柚皮苷可清除活性氮并抑制线粒体自噬。因此我们提出活性氮诱导过度线粒体自噬造成脑缺血再灌注损伤，而柚皮苷可抑制该过程达到神经保护作用的假说。本项目拟从三个层次验证假说：采用药物及基因干预手段说明过度线粒体自噬在脑缺血再灌注损伤的作用；利用活性氮供体及抑制剂阐释活性氮与PINK1/Parkin通路介导的线粒体自噬的关系；以柚皮苷为例说明针对活性氮介导的过度线粒体自噬是抗脑缺血再灌注损伤的治疗新策略。

线粒体自噬是脑卒中研究新方向，脑缺血再灌注产生大量自由基诱导线粒体自噬。通过自噬清除受损线粒体维持细胞稳态，而过度线粒体自噬则会导致细胞死亡。活性氮是重要的自由基，在线粒体损伤过程中起关键作用，目前有关活性氮调控脑缺血再灌注损伤中线粒体自噬的研究仍是空白。本项目旨在探索脑缺血再灌注损伤中，过度线粒体自噬对于该病理过程影响及其调控机制。研究结果表明：脑缺血再灌注过程中线粒体自噬与大脑损伤有密切关系。缺血再灌注损伤后过氧亚硝基水平及线粒体自噬程度均明显提高，且过氧亚硝基解构剂FeTMPyP干预后，通过减少脑内过氧亚硝基水平，进而抑制线粒体自噬，减轻大脑缺血再灌注损伤，其机制与抑制PINK1/Parkin介导的线粒体自噬活化，避免LC3向线粒体聚集，同时抑制了动态相关线粒体蛋白Drp1从细胞质向线粒体的转移有关。而柚皮苷可通过清除一氧化氮及超氧阴离子，抑制过氧亚硝基活性，避免大脑线粒体自噬水平过度激活，从而保护大脑缺血再灌注损伤。由于柚皮苷能够通过血脑屏障，直接作用于缺血再灌注大脑，抑制Parkin向线粒体转移，降低LC3II/I比例，从而抑制缺血再灌注损伤诱导的线粒体自噬。通过本研究，我们以柚皮苷为例说明针对活性氮介导的过度线粒体自噬是抗脑缺血再灌注损伤的治疗新策略。