循环mtDNA-NLRP3炎性体信号通路介导创伤性急性肺损伤发生的分子机制

The upstream mechanism of systemic inflammatory response syndrome(SIRS) has become one of the most important breakthroughs in the basic research, early diagnosis and treatment of post-traumatic acute lung injury (ALI). Recent studies demonstrated that mitochondrial DNA (mtDNA) released from mitochondrion into cytoplasm directly bound to and activated NLRP3 inflammasome and promoted local inflammation. Our previous study found that circulating mtDNA concentration was increased significantly in patients diagnosed as post-traumatic ALI and the elevated concentration was positively correlated with the severity of disease, furthermore, the mtDNA exposure would up-regulate NLRP3 mRNA and increase the expression of IL-1β in cultured THP-1 cell. Thus, it is hypothesized that circulating mtDNA activate NLRP3 inflamasome and contribute to the onset and development of post-traumatic ALI. This project aims to identify the molecular mechanism of mtDNA-NLRP3 inflamasome signaling pathway contributing the development of SIRS and the onset of post-traumatic ALI, and to evaluate the predictive and prognostic value of mtDNA and NLRP3 downstream inflammatory cytokines by monitoring the dynamic changes of abovementioned factors in post-traumatic ALI patients . Thus, this project is designed to unravel a new mechanism of mtDNA promoting post-traumatic ALI and probably provide a theoretical basis for the early intervention strategy targeted mtDNA-NLRP3 signal in post-traumatic ALI.

全身炎症反应综合征（SIRS）的上游机制是创伤性急性肺损伤（ALI）基础研究和早期诊治的重要突破点之一。研究显示线粒体DNA（mtDNA）由线粒体释放入细胞质后可与NLRP3结合，激活NLRP3炎性体促进局部炎症反应。本课题组前期研究发现：循环mtDNA水平在创伤性ALI患者明显升高且与病情相关；外源性mtDNA可上调THP-1细胞NLRP3 mRNA、增加IL-1β蛋白表达。我们提出假说：循环mtDNA激活NLRP3炎性体信号通路并促进创伤性ALI发生、发展。本项目拟明确循环mtDNA-NLRP3炎性体信号通路促进SIRS、启动创伤性ALI的分子机制，监测创伤性ALI患者的循环mtDNA和NLRP3炎性体下游炎性细胞因子动态变化，以明确其在创伤性ALI发生及预后中的预警作用。为阐明循环mtDNA启动创伤性ALI发病新机制及针对循环mtDNA-NLRP3信号通路的早期干预策略提供理论依据。

损伤相关分子模式（damage associated molecular patterns，DAMPs）介导的无菌性炎症反应是无菌性急性肺损伤的重要原因。创伤患者外周血血浆中线粒体DNA（mitochondrial DNA，mtDNA）显著增加，并且可导致模式动物肺组织损伤和过度炎症反应。除Toll样受体外（toll-like receptors，TLRs），NOD样受体（NOD-like receptors，NLRs）是过度炎症反应的关键分子。本课题的研究显示，细胞外mtDNA可促进THP-1巨噬细胞IL-1β和IL-18产生增加，培养上清中caspase-1活性和caspase-1 p20活性亚单位蛋白含量均显著增加。细胞外mtDNA促进THP-1巨噬细胞NLRP3、AIM2、ASC和caspase-1转录增加，同时可促进NLRP3、procaspase-1表达增加和p38 MAPK激活、NF-κB核转移。Caspase-1 siRNA和NLRP3-siRNA均可抑制细胞外mtDNA相关IL-1β产生，而AIM2-siRNA则对此无显著影响。采用TLR9-siRNA抑制TLR9的表达后，细胞外mtDNA介导的NLRP3表达显著下降；分别采用p38-siRNA和SB203580抑制p38 MAPK表达和活性，采用PDTC抑制NF-κB激活，结果显示抑制p38 MAPK和NF-κB后NLRP3转录和表达均显著下降。体内实验显示，mtDNA经气管插管局部给药可成功诱导C57BL/6小鼠肺湿干比升高，肺组织水肿、肺泡壁增厚、炎症细胞浸润等炎症反应性形态学改变和肺泡上皮细胞凋亡。mtDNA经气管插管局部给药可促进C57BL/6小鼠支气管肺泡灌洗液中IL-1β、IL-18浓度和caspase-1活性均显著升高。小鼠肺组织NLRP3转录和表达均显著增加，肺组织caspase-1 p20亚单位蛋白含量也显著增加。Belnacasan (VX-765)抑制caspase-1活性后可减轻上述损伤和炎症反应。综上所述，我们得出结论：细胞外mtDNA可通过TLR9-p38 MAPK促进NF-κB核转移，介导NLRP3炎性体组件的表达增加，诱导细胞内NLRP3炎性体激活和IL-1家族细胞因子合成和释放，该过程可诱导小鼠肺组织过度炎症反应和肺泡上皮细胞凋亡，促进肺损伤发生。