线粒体自噬障碍致“mtDNA逃逸”介导慢性危重症肠黏膜免疫麻痹的研究

Chronic critical illness (CCI) is a new-onset manifestation parallel with the development of intensive care medicine, of which the pathogenesis remains to be elucidated. Bacterial translocation plays as direct lethal factor in CCI. Mitochondrial DNA (mtDNA) released during early phase of critical illness induces significant damage to organ functions. We found that mtDNA was accumulated in tissue and released into blood accompanied with mitophagy dysfunction in CCI patients. We further hypothesize that mtDNA released from damaged intestinal epithelial cell could be recongized by mucosal dendritic cells to activate mitophagy. Multiple autophagy-associated factors inhibit cGAS-STING pathway that is assumed to be activated by mtDNA, leading to suppressed production of inflammatory mediators. MtDNA escaped from impaired mitophagy reciprocally promotes mitophagy dysfunction in a "vicious self-cycle" manner which induces mucosal immune paralysis. We will utilize bacterial typing methods including PFGE, MLST and MALDI-TOF MS to prove and evaluate bacterial translocation in CCI. We aim to elucidate underlying mechanism by which impaired mitophagy induces "mtDNA escape" and contributes to mucosal immunoparalysis in CCI. We expect to provide new strategies for the management and prevention of chronic critical illness.

慢性危重症是重症医学发展中新出现的疾病表现形式，其发病机制亟待阐释。肠源性感染带来的再次与多次打击往往是慢重症致死的直接因素。疾病早期大量释放的mtDNA可介导器官功能继发性损害。本团队前期研究发现慢重症患者存在mtDNA大量堆积及线粒体自噬障碍。本项目提出感染与创伤等应激引起肠黏膜损伤并释放大量mtDNA，被肠黏膜树突状细胞感知并捕获，激活线粒体自噬；多种自噬关键因子抑制本应被mtDNA激活的cGAS-STING通路，导致炎性介质无法生成；同时慢重症时线粒体自噬的融合及降解障碍引起“mtDNA逃逸”并大量堆积，以“恶性自循环”方式加重线粒体自噬障碍，诱导慢重症肠黏膜免疫麻痹；基于“肠唇状瘘”模型，利用细菌溯源技术直接证实肠道菌群可通过肠黏膜诱发肠源性感染。本研究拟阐述线粒体自噬障碍致“mtDNA逃逸”介导慢重症肠黏膜免疫麻痹的机制，为慢重症的防治提供新的靶点与干预途径。

脓毒症是一种机体对感染的免疫反应失调所引起的危及生命的器官功能障碍，后期可发展为慢性危重症，伴随肺脏、肝脏、胃肠道、肾脏等多器官的功能障碍。目前认为，多器官功能障碍是由于微生物代谢产物或细胞损伤释放的损伤相关分子模式（DAMPs）激活实质细胞以及免疫细胞，导致氧化应激和炎症反应增强所致。然而，细胞激活并导致器官损伤的机制尚未阐明，这限制了有效治疗措施的发展。本项目通过收集慢性危重症病人血液样本，结合临床资料，利用ELISA、免疫印迹等方法发现循环DAMPs水平与疾病严重程度相关；利用盲肠结扎穿孔以及LPS腹腔注射等多种模型模拟危重症，以验证临床假设；此外，通过构建STING敲除小鼠，使用免疫荧光、实时定量PCR、免疫印迹等分子生物学实验，探究STING信号通路在疾病中的作用机制；并创新采用线粒体DNA（mtDNA）腹腔注射模型，验证以mtDNA为代表的循环DAMPs对STING信号通路的激活在疾病中的作用；进而通过体内体外实验，利用自噬流检测、荧光共定位、免疫印迹等方法，发现STING过度激活可导致溶酶体酸化障碍，影响自噬，进一步促进STING激活；最后回归临床研究，探索慢性危重症患者继发感染病原菌的来源。.本项目阐明了以mtDNA为主的循环DAMPs导致慢性危重症多脏器功能损伤的病理生理机制，揭示了mtDNA激活STING通路诱发免疫应答，过度激活的STING可通过干扰溶酶体酸化影响自噬的降解，进一步加重炎症反应；基于“肠唇状瘘”模型，利用细菌溯源技术直接证实肠道菌群可通过肠黏膜诱发肠源性感染。本项目的研究结果为危重症相关并发症的发生发展机制提供了深入方向，为提高慢性危重症治疗效率、改善临床预后提供了明确目标。