TLR4/HIF-1α干预对创伤失血性休克后急性肺损伤的作用及机制研究

Mortality associated with acute lung injury (ALI) induced by trauma hemorrhagic shock (T/HS)is very high due to the shortage of effective interfering choice.Ischemia/reperfusion insult to gut,uncontrolled pulmonary inflammatory response and hypoxia in microenviroment are recognized as the critical triggering and deteriorating mechanisms for ALI induced by T/HS.We found that inflammatory factors tumor necrosis factor(TNF)-α and interleukin (IL)-4 can activate HIF-1α,and there is a synergic effect with hypoxia;furthermore, we found that silencing of TLR4 gene can ameliorate ALI induced by endotoxin, and inhibition of HIF-1α activity alleviates ALI induced by T/HS.In our pilot test, we found significant activation of TLR4/HIF-1α during ALI induced by T/HS.Thus, we suppose interfering TLR4/HIF-1α is a new effective therapy choice for ALI by T/HS. We plan to start from the critical triggering and deteriorating mechanisms of ALI induced by T/HS to explore the effects and mechanisms of intereing TLR4/HIF-1α on ALI by T/HS. We will set up T/HS animal and cell model, using gene engineering, RNA interference and report gene assay methods to validate our assumption from whole-cell-transcription factor facets. Conclusions from our study will provide new theory and interfering targets for the treatment of ALI induced by T/HS.

创伤失血性休克（T/HS）后急性肺损伤（ALI）由于缺乏有效干预手段而死亡率很高。肠道缺血再灌注损伤和肺部失控性炎症反应、微环境缺氧分别是 T/HS后 ALI的关键启动机制和恶化机制。我们发现炎症因子TNF-α和IL-4可激活HIF-1α，且和缺氧有协同效应；进一步研究证实，沉默TLR4基因可改善内毒素所致ALI，且阻断HIF-1α活性也可改善T/HS后ALI。我们的预实验结果提示TLR4/HIF-1α在T/HS后ALI过程中明显激活。因此，我们推测：干预TLR4/HIF-1α是有效防治T/HS后ALI的新靶点。我们拟从T/HS后ALI的关键启动机制和恶化机制入手，制备T/HS后ALI动物模型和细胞模型，应用基因工程、RNA沉默、报告基因分析等技术，从整体-细胞-转录因子层次明确干预TLR4/HIF-1α对T/HS后ALI的作用及机制，研究结果将为防治 T/HS后ALI提供新思路和新靶点。

创伤失血性休克（T/HS）后急性肺损伤（ALI）由于缺乏有效干预手段而死亡率很高。核转录因子HIF-1α是机体缺氧/炎症状态下的关键转录因子，TLR4是组织细胞损伤时感受损伤信号的重要受体之一。我们的前期研究发现局部阻断HIF-1α激活可以改善大鼠创伤失血性休克后的肺损伤程度。在本研究中我们发现在TLR4突变型和野生型小鼠输注T/HS或T/SS淋巴后，肺损伤指标如Evans blue，肺干/湿比，MPO水平以及肺病理损伤分级等发现TLR4突变型小鼠输注T/HS淋巴后肺损伤情况较野生型明显减轻，且HIF-1α在肺组织的表达水平也较TLR4野生型明显减少，体外培养TLR4突变型和野生型小鼠肺血管内皮细胞后采用RNAi干扰TLR4表达后发现HIF-1α基因表达有明显下降。同时阻断NF-κB信号途径可阻断T/HS淋巴液对HIF-1α的诱导作用。我们也研究了补体C5a在肠道缺血/再灌注损伤后的肺损伤中的地位。在肺巨噬细胞，IR后肺损伤时C5aR表达明显升高，且C5a与其受体的结合可导致肺巨噬细胞的凋亡。采用atg5基因敲除小鼠建立IR后肺损伤模型发现，C5a是通过介导bcl2的降解，导致肺巨噬细胞自噬过程加强引起细胞凋亡的。在麻醉学科，异氟烷是常用的吸入麻醉药。在内毒素导致的急性肺损伤过程中，异氟烷可以改善肺损伤程度，激活HIF-1α-HO-1信号途径，降低miR-155的表达，且过表达miR-155可阻断异氟烷对HIF-1α-HO-1信号途径的激活作用。因此，本研究发现T/HS后肺损伤的发生机制中有TLR4介导转录因子HIF-1α的活化过程参与，C5aR介导的肺巨噬细胞自噬过程在IR后肺损伤发生过程中发挥重要作用，且异氟烷对肺损伤的后处理效应的分子机制有miR-155-HIF-1α信号途径的参与，本研究为创伤后肺损伤的发生机制提供了新的思路，为治疗肺损伤的发生提供了新的治疗靶点。