线粒体自噬抑制肺泡巨噬细胞焦亡阻断mtDNA对脓毒症肺损伤放大作用的实验研究

It is well known that mitochondrial in alveolar macrophages (AM) are damaged and release mitochondria DNA (mtDNA) to cytoplasm in sepsis-induced lung injury, lead to production of caspase-1, cleavage the precursor of IL-1β into mature IL-1β, Recent studies have shown that caspase-1 also induces cell death in a variety of cells, causing the release of inflammatory factors such as IL-1β, and amplifying the inflammatory response. However, whether mtDNA in AM can induce pyroptosis has not been reported. In our exploratory study (i.e., the AM treated with LPS and mtDNA) we found that IL-1β and pyroptosis proteins, GSDMD, were higher in expression levels than those in AM stimulated by LPS; Other studies have been confirmed by the fact that when mitochondria are damaged, Parkin would be recruited to the mitochondrial outer membrane to initiate mitophagy. Therefore, we propose a scientific hypothesis that enhancing mitophagy in sepsis-induced lung injury, can reducing the release of mtDNA in AM, inhibit pyroptosis. In the proposed project we plan to construct a lentiviral vector to transfect pakin gene to upregulate mitophagy and knocked out the GSDMD pyroptosis gene in sepsis-induced lung injury models in vitro and in vivo, to study the effects and mechanisms of its impact on inhibition of pyroptosis and block the amplification effect of mtDNA on the pulmonary inflammatory. We anticipate that the proposed research will provide a new way to treat septic lung injury.

脓毒症肺损伤时肺泡巨噬细胞（AM）线粒体受损并向细胞质中释放出线粒体DNA（mtDNA），引起caspase-l生成，将IL-1β前体剪切为成熟IL-1β。最新研究表明，在多种细胞中caspase-l同时还诱导焦亡，引起IL-1β等炎症因子释放。然而，AM内mtDNA能否诱导细胞焦亡未见报道。我们探索性采用LPS联合mtDNA作用于AM，发现IL-1β、焦亡蛋白GSDMD均较LPS组升高；另有研究证实，线粒体受损时Parkin被招募至线粒体外膜可启动线粒体自噬，清除受损线粒体。因此，我们提出科学假说，脓毒症肺损伤时，增强线粒体自噬，可清除受损线粒体，降低AM内mtDNA释放，抑制焦亡。本项目拟在脓毒症肺损伤体内外模型中，构建慢病毒载体上/下调pakin基因调控线粒体自噬，并敲除GSDMD焦亡基因，研究其对抑制焦亡、阻断mtDNA对肺内炎症放大作用的影响及机制，为治疗脓毒症肺损伤提供新途径。

线粒体自噬是清除受损或功能障碍线粒体的自噬过程，对维持细胞内环境的稳态具有重要意义。我们前期研究发现，在脓毒症肺损伤中，肺泡巨噬细胞(AM)线粒体受损释放出线粒体DNA（mtDNA），能够诱导细胞焦亡，放大肺内炎症反应。然而线粒体自噬能否调控细胞焦亡，阻断mtDNA对炎症反应的放大作用未见报道。本项目在脓毒症肺损伤体内外模型中，外源性予以mtDNA刺激，观察mtDNA对细胞焦亡及炎症因子水平的变化规律，随后调控线粒体自噬，观察线粒体自噬对脓毒症肺损伤的影响，最后进一步从miRNA表观遗传学的角度，探索了线粒体自噬调控AM细胞焦亡的分子机制。结果发现：（1）mtDNA通过NLRP3炎症小体通路，诱导AM焦亡，放大脓毒症肺内炎症反应；（2）增强线粒体自噬可抑制细胞焦亡，减轻mtDNA对脓毒症肺损伤的放大作用；（3）增强线粒体自噬可降低胞质mtDNA水平，使miR-138-5p启动子去甲基化，从而抑制NLRP3炎症小体活化及AM焦亡，减轻脓毒症肺损伤。基于以上结果，我们得出结论，增强线粒体自噬可减少mtDNA释放，抑制NLRP3炎症小体活化及AM焦亡，降低肺内炎症反应，对脓毒症肺损伤发挥保护作用，为临床治疗脓毒症肺损伤提供了新路径。