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

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。

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.