HMGB1-PAD4信号促进中性粒细胞外捕获网形成在肠缺血再灌注致急性肺损伤中的作用及机制研究

Acute lung injury (ALI) induced by intestinal ischemia/reperfusion (I/R) injury is one of the leading causes of death in critical ill patients, but the mechanisms are still not completely understood. Activated neutrophils have been recently described to form neutrophil extracellular traps (NETs) in response to various pathogenic infections. However, overproduction of NETs have also been implicated as harmful contributors in inflammatory conditions. Previous researches already showed that NETs formation can occur dependently on necroptosis signaling pathway. Meanwhile, we also found that HMGB1 released after intestinal I/R induced epithelial necroptosis led to the activation of TLR4 signaling. Our preliminary study showed that a large amount of NETs formed in the lung tissue after the inflammation storm induced by intestinal I/R, and DNase 1 treatment resulted in significantly less NET formation and lung injury alleviation. Moreover, the key enzyme PAD4 in NETs formation can be regulated by recombinant HMGB1 in a concentration-dependent manner in vitro. Therefore, we hypothesized that HMGB1 released from intestinal I/R-induced epithelial necroptosis led to the PAD4 enzyme activation, which further mediates NETs formation and promotes ALI after intestinal I/R. By implying in vivo and in vitro models, the current research is designed to explore the underlying new mechanisms of neutrophil mediated intrinsic immune dysfunction in ALI after intestinal I/R.

肠缺血再灌注（I/R）后的急性肺损伤（ALI）是导致危重患者死亡的重要原因，但调控机制尚不明确。炎症时中性粒细胞（PMN）激活后被证实能形成中性粒细胞外捕获网（NETs），虽可发挥抗感染功能，但过度形成的NETs却导致自身损伤。已知NETs可继发于程序性细胞坏死。我们前期证明肠I/R致肠上皮细胞程序性坏死后释放HMGB1促使胞内TLR4信号激活。预实验显示肠I/R后炎症风暴致肺内激活的PMN形成NETs，而消除NETs能减轻肺损伤；同时HMGB1能调控NETs关键酶PAD4的表达。由此我们推测HMGB1通过调控PAD4活化促进肺内NETs形成，这种功能失衡介导了ALI的发生发展。本项目拟通过一系列实验证实肠I/R后HMGB1与PMN表面TLR4受体结合调控PAD4活性并激活PMN过度形成NETs，促进肺的炎性损伤和局部免疫功能障碍；最终从PMN固有免疫失调角度揭示肠I/R致ALI的新机制。

肠道缺血再灌注（Ischemia/reperfusion，I/R）损伤是围术期及临床急危重症疾病中常见的病理生理过程。在危重患者中，肺脏常是肠道I/R后第一个衰竭的远程器官。大量中性粒细胞聚集活化所致的失控性炎性反应被认为是各种病因导致肺损伤的根本原因。我们通过动物实验发现小鼠肠I/R后可导致肺组织呈动态损伤，该损伤与肠道缺血后聚集于肺组织中的中性粒细胞激活形成中性粒细胞外捕获网（NETs）相关，使用PAD4抑制剂抑制或DNase I酶降解NETs的治疗措施均可显著减轻肠I/R后急性肺损伤。探索其机制表明:肠I/R后肠黏膜上皮细胞发生程序性坏死Necroptosis，其释放的HMGB1分子及其下游Myd88信号通路促进肺组织中性粒细胞激活并形成NETs加重急性肺损伤。.同时，肠道作为多器官功能不全综合症的“启动”和“枢纽”器官，肠I/R损伤后导致肠外其他器官损伤的机制尚不清楚。我们进一步通过动物实验发现：①肠道I/R损伤可以导致急性肝脏损伤，以再灌注后6小时最为显著。肠道大肠杆菌Escherichia coli肝脏内异位、肝细胞中HMGB1从胞核向细胞质内移位、肝细胞发生Necroptosis、肝脏库普弗细胞和循环巨噬细胞向M1型巨噬细胞极化等机制介导了上述的病理生理过程。②肠I/R后可造成远程胰腺组织的损伤，该过程与肠黏膜上皮细胞和胰腺腺泡细胞均发生Necroptosis相关。③肠I/R后黏膜上皮细胞释放的HMGB1通过PERK-ATF4-CHOP通路激活肾脏细胞内质网应激介导了肾功能损伤。上述研究结果系统性探索了肠I/R损伤后导致肠外多器官损伤的相关机制并提出了可能的干预措施。