肺泡巨噬细胞焦亡在失血性休克后急性肺损伤中的作用及机制研究

Trauma/hemorrhagic shock (HS) often renders patients more susceptible to a secondary stimulus (e.g., infection) resulting in development of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) by activating and priming the inflammatory process. The underlying mechanism of how HS leads to SIRS and MODS has yet to be fully determined. Acute lung injury (ALI) is a major component of MODS and often serves as a direct cause of patient death. Our long-term goal is to determine the mechanism by which trauma/HS promotes ALI, thereby, potentially identifying novel targets for prophylactic intervention. Inflammation is a dynamic process that exhibits an auto-regulatory nature. Through the auto-regulation, adaptive immunity can be activated and resolution of the inflammation occurs following the initiation and development of inflammation. In trauma/HS patients, enhanced inflammation and increased susceptibility to infection suggest that there are as yet unclear mechanisms that cause immune dysregulation and consequently prolong and exacerbate the inflammatory process. We now demonstrate in our preliminary studies that in a mouse HS/trauma (HS/T) model, HS induces AM pyroptosis, a caspase-1-dependent programmed cell death, in a late phase of inflammation (20 h after HS); and this HS-induced pyroptosis is mediated by a novel signaling pathway, in which receptor for advanced glycation end products (RAGE)-dependent endocytosis of HMGB1 activates pyroptosome assembly and cell pyroptosis, and in which inflammasome activation primes for the cell pyroptosis. Our further observations suggest that induction of AM pyroptosis enhances inflammation by releasing or promoting healthy AM to release pro-inflammatory cytokines and chemokines, augmenting polymorphonuclear neutrophil (PMN) chemotaxis, and suppressing T lymphocyte (T-cell) migration. Moreover, we have also shown in our previous and preliminary studies that HS enhances HMGB1/TLR4-induced upregulation of TLR2 in AM, which in turn augments AM pyroptosis in response to TLR2 ligands. Based on the above findings, we hypothesize that: 1) AM pyroptosis promotes the development of ALI after HS through influencing the inflammatory process; 2) HMGB1-RAGE signaling serves as a novel mechanism that induces AM pyroptosis in HS; and 3) HMGB1-upregulated TLR2 serves as an important mechanism for mediating HS-primed AM pyroptosis. In order to test these hypotheses, we propose the following three specific aims: Specific Aim #1. To determine the role of AM pyroptosis in the development of ALI following HS. Specific Aim #2. To determine the molecular mechanism through which HS induces AM pyroptosis. Specific Aim #3. To determine the mechanism of HS-primed AM pyroptosis in response to TLR2 activation.

失血性休克（HS）是临床上常见重症，常起因于创伤、手术导致的大出血。患者复苏一至二周后后常面临二次死亡峰期，即发生全身炎症反应综合征（SIRS）以及多器官功能障碍综合征（MODS），占ICU病人死因的50%以上。急性肺损伤（ALI）是MODS的主要组成部分，也是患者的最直接死因。HS复苏后一周内是防治SIRS和ALI的最佳时期。但HS导致SIRS-MODS的机制尚不清楚，临床缺乏干预靶点，国际上尚无遏制患者进入二次死亡峰期的有效措施。我们的前期研究发现HS后内源性损伤相关分子模式分子HMGB1通过诱导其本身在肺泡巨噬细胞的内化和一条新的细胞内信号通路导致肺泡巨噬细胞焦亡，进而深度影响肺炎症过程，促进ALI的形成。本研究中我们将进一步从分子、细胞、动物及临床水平研究肺泡巨噬细胞焦亡在创伤与HS所致的ALI中的作用和机制，为有效遏制HS后患者进入SIRS-MODS阶段提供干预治疗靶标。

失血性休克（HS）是临床上常见的病症，常起因于创伤，手术导致的大出血。HS 患者复苏后的一至二周内常面临着第二次死亡峰期，多由于发生全身炎症反应综合征（SIRS）。HS 患者中SIRS 的发生率高达43.2%，并可进一步发展为多器官功能障碍综合征（MODS）。急性肺损伤（ALI）是MODS 的主要组成部分，也是导致病人死亡的直接原因之一。在HS复苏后的一周内，是临床上干预防治SIRS和ALI的最佳时期。但HS导致SIRS-MODS 的机制尚不清楚，临床上缺乏干预靶点，目前尚无遏制患者进入第二次死亡峰期的有效措施。本项目的主要研究结果：1）通过对 WT，RAGE-/-和Nlrp3-/-小鼠，及体外分离的肺血管内皮细胞（MLVEC）的研究发现，HS通过HMGB1-RAGE信号通路增加LPS诱导的MLVEC焦亡；2）通过对WT小鼠，及WT，TLR4-/-和RAGE-/-小鼠分离的MLVEC研究发现，HS通过ROS-TXNIP通路增强LPS的致Nlrp3焦亡小体的激活作用；3）采用体外分离的WT，RAGE-/-和TLR4-/-小鼠的MLVEC研究发现，HMGB1通过RAGE和dynamin依赖的信号通路发生内吞作用；4）内吞的HMGB1致溶酶体破裂和Cathepsin B激活，并促进焦亡小体的形成，Caspase-1的活化和IL-1β的释放。本研究初步证实，HMGB1 通过RAGE和dynamin 依赖的信号通路启动MLVEC内吞HMGB1；内吞的HMGB1 随后触发一系列细胞内的分子效应，包括溶酶体破裂，CathepsinB的释放与活化，焦亡小体的形成和Caspase-1 的激活，最终导致MLVEC的焦亡。通过本课题的研究，我们基本明确了MLVEC焦亡在HS后ALI发生发展中的重要作用及机制，阐明了HS诱导MLVEC焦亡的分子机制，为临床干预防治HS后ALI提供了靶点及理论依据。