MTDNA/NLRP3活化切割GSDMD蛋白介导肺巨噬细胞焦亡在ALI中的作用机制

Accumulating evidences suggest that GSDMD was identified as a caspase substrate and a key component of NLRP3 inflammasome. Cleavage of GSDMD could initiate inflammatory caspases then trigger cell pyroptosis which causing massive leakage of cytosolic contents and eventually greatly augmented inflammation. Our previous study demonstrated that stimulation by lipopolysaccharide (LPS) could induce the release of critical proinflammatory cytokines that are necessary to mediate cell necrosis and mtDNA escaping. However, whether mtDNA escaping could induce cell pyroptosis and eventually lead to ALI? Its mechanism has not yet been fully elucidated. So we proposed that mtDNA escaping autophagy activate NLRP3 via TLRs/MyD88/NF-κB signaling pathway to cleave GSDMD, this in turn activate Caspase-1 which mediate cell pyroptosis, then lead to SIRS, and ultimately ALI. To address the hypothesis, through founding animal and cell models as well as using fluorescence RT-PCR, Western blot, mass-spectrometric, gene silencing technique et.al, from the overall of the molecules, cells, tissues and animals level to elucidate the mechanism of NLRP3 activation by mtDNA. Our study not only focus on the mechanism of GSDMD cleavage by NLRP3 but also to explore the mechanism of Caspase-1 mediating alveolar macrophages autophagy and pyroptosis. This study will provide a new insight for the pathogenesis of ALI by autophagy and pyroptosis, and a new point in prevention and treatment of ALI.

研究表明，GSDMD是NLRP3关键组成蛋白，Caspase的底物，切割GSDMD可激活炎性Caspase引发细胞焦亡,释放大量细胞内容物，放大炎症反应。前期研究发现，内毒素刺激可诱发大量炎性因子释放,导致细胞变性坏死并伴有mtDNA逃逸，然而，逃逸的mtDNA是否引发细胞焦亡导致ALI发生？尚未见报道。因此，我们提出假说：自噬逃逸mtDNA通过TLRs/MyD88/ NF-κB信号通路活化NLRP3切割GSDMD蛋白激活Caspase-1介导细胞焦亡，诱发SIRS，发生ALI。为验证假说，通过动物及细胞模型，采用qRT-PCR、WB、质谱技术、基因沉默等，从分子、细胞、组织及动物整体水平探讨mtDNA活化NLRP3作用机制；探讨NLRP3切割GSDMD分子机制；探讨Caspase-1介导肺泡巨噬细胞自噬与焦亡作用，从自噬与焦亡新视点揭示ALI的发病机制，为ALI治疗寻找靶点提供新思路。

利用LPS刺激，建立小鼠 ALI 模型并验证成功及体外肺巨噬细胞模型构建，应用Real time PCR、Western blot、慢病毒载体转染、基因沉默等技术手段，从分子、细胞、组织以及动物水平等层次围绕以下目标进行研究：ALI 中肺巨噬细胞自噬及线粒体ROS、DNA 逃逸(mtROS、mtDNA)机制；探讨自噬逃逸线粒体mtROS、mtDNA通过TLR9/ myd88/NF-κB信号通路调控NLRP3炎性小体活化介导巨噬细胞焦亡的机制；探讨活化NLRP3炎性小体激活Caspase-1切割焦亡蛋白GSDMD介导肺巨噬细胞焦亡，最终焦亡肺巨噬细胞致使肺上皮细胞焦亡导致肺损伤的作用机制。.结果：LPS诱导小鼠肺组织炎症反应；活化NLRP3炎性小体；发生肺巨噬细胞自噬；溶酶体耗竭导致mtROS、mtDNA逃逸；mtROS、mtDNA促进肺部炎症反应，且通过TLR9/MyD88/NF-κB通路激活NLRP3炎性小体活化Caspase1切割焦亡蛋白GSDMD介导肺巨噬细胞焦亡；活化Caspase1介导肺泡巨噬细胞焦亡激活NLRP3/Caspase1/ GSDMD导致肺上皮细胞焦亡，促进炎性的级联反应，最终导致肺损伤的发展。.本项目从细胞自噬与细胞焦亡的新视点揭示ALI的发病机制，为ALI防治寻找治疗靶点提供新思路。.本项目培养了一名博士研究生及一名硕士研究生，培养了三名技术骨干考取了博士研究生；在此基础上，获得国家基金委资助项目1项，广西重大研发项目2项；并在学术会议进行交流及推广应用；撰写了三篇高质量的学术论文。学科获得了广西麻醉学临床研究中心，在一定程度上推动了学科的发展。. 存在问题：内源性mtDNA的来源研究并未深入，没有直接证据表明Caspase1与GSDMD的关键作用，没有直接利用基因敲除小鼠开展实验研究。因此，仍需进一步加深研究，为ALI发生机制提供更可靠证据。