NETs诱导肺泡上皮细胞焦亡在急性肺损伤中的意义及机制研究

Acute lung injury (ALI) is a complex and severe disorder of the lungs and remains a leading cause of death in the Intensive Care Unit. Dysregulated inflammation, inappropriate accumulation of inflammatory mediators, uncontrolled activation of inflammatory effector cells remain central pathophysiologic concepts in ALI. Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA from neutrophils, which kill or suppress fungal and bacterial proliferation. Pyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. Both NETs and alveolar epithelial cell (AEC) pyroptosis are playing important roles in regulating the inflammatory process after infection. However, the effect of NETs and AEC pyroptosis on the pathogenesis of ALI remains unclear. Our preliminary data demonstrated that 1) Pyroptosis occurred in AEC during LPS-induced ALI in mice; 2) NETs and HMGB1 were released into bronchoalveolar lavage fluid during LPS-induced ALI and reached peak concentrations at 6 hours after LPS delivery; 3) Inhibition of NETs formation alleviated the pyroptosis of AEC in LPS-induced ALI. Based on those data we propose that LPS-induced PMNs migrate into the alveolar airspaces followed by NETs formation and HMGB1 release from NETs. NETs-derived HMGB1, acting through RAGE-dependent signaling, triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by caspase-1 and caspase-11 activation, which ultimately leads to pyroptosis in AEC.Our study demonstrates the underlying mechanisms by which NETs induces pyroptosis in AEC both in vitro and in vivo, providing evidence that therapeutic intervention to modulate the NETs formation and pyroptosis in AEC may be an effective strategy to promote the resolution of ALI.

肺内过度炎症反应及组织损伤是急性肺损伤(ALI)的本质表现。中性粒细胞胞外陷阱(NETs)是中性粒细胞(PMN)受到刺激后形成的网状结构，肺泡上皮细胞(AEC)焦亡是一种致炎的细胞死亡方式，两者均能调控感染后的炎症过程，但其在ALI发生发展中的作用尚未明确。我们的前期研究显示：①在LPS诱导的ALI小鼠模型中，AEC发生焦亡②肺泡灌洗液中NETs和HMGB1的含量在给予LPS6小时后达峰③抑制肺泡腔内NETs形成能减轻AEC焦亡。我们推测：LPS诱导PMN迁移至肺泡腔形成NETs，并伴随大量HMGB1释放，NETs源性HMGB1激活AEC上RAGE信号通路，随后触发胞内一系列分子效应，包括CatB的活化和释放、ASC的合成、Caspase-1/Caspase-11的活化，最终导致AEC焦亡。本课题拟从不同层次探讨ALI时NETs诱导AEC焦亡的机制，以期为临床上有效干预ALI提供新的靶点。

急性肺损伤（ALI）是临床上常见的危重症之一，其本质是肺内失控性炎症反应及组织损伤。中性粒细胞胞外陷阱（NETs）是PMN受到刺激后将自身染色体进行解压缩并释放到细胞外形成的网状结构，其形成过程中会伴随大量物质释放。肺泡上皮细胞（AEC）焦亡是一种致炎的细胞死亡方式，两者对感染后的炎症过程均有调控作用。但NETs是否能够通过诱导AEC焦亡参与ALI目前尚不清楚。本项目主要研究结果显示：1）LPS可以时间依赖性诱导小鼠AEC焦亡，于18h达峰；2）LPS通过Caspase-1和Caspase-11诱导AEC焦亡，进而释放炎症因子，引起肺组织损伤；3）LPS呈时间依赖性诱导小鼠肺泡腔内NETs的形成，于6h达峰；4）体内外产生的NETs均能够诱导小鼠AEC焦亡及炎症介质释放增加；5）TLR4基因敲除及抑制NETs的形成均能够降低AEC的焦亡，减轻肺组织损伤；6）体外实验证实NETs形成过程中伴随HMGB1的释放，于2 h达峰；7）小鼠肺泡腔内NETs形成过程中伴随HMGB1的释放，于6h达峰；8）分别采用WT、HMGB1-/-小鼠和RAGE-/-小鼠构建ALI模型，证实NETs源性HMGB1和RAGE在诱导AEC焦亡中发挥了重要作用。本研究结果初步证实：LPS诱导PMN 迁移至肺泡腔内并使其活化形成NETs，NETs 释放的过程中伴随HMGB1的释放，NETs源性HMGB1 通过激活AEC的RAGE信号通路，触发细胞内一系列分子效应，并通过对下游Caspase-1/ Caspase-11 进行剪切和活化，最终导致AEC 焦亡，而AEC 的焦亡进一步加重炎症反应和对感染的敏感性。本项目通过探讨ALI时NETs诱导AEC焦亡的意义和分子机制，以期为临床上有效干预ALI提供新的靶点。