PAI-1/PECAM/p38MAPK/PI3K通路在小鼠肺损伤中调控中性粒细胞肺内募集与活化机制的研究

Neutrophils, which were regulated by fibrinolysis and coagulation system, played a fundamental role in the pathogenesis of acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). However, the mechanism of recruitment and activation of neutrophils by fibrinolysis and coagulation system was not elucidated. The plasminogen activator inhibitor-1 (PAI-1), an important member of fibrinolysis and coagulation system, was positively correlated with mortality in patients with ARDS/ALI. In early phase of ARDS/ALI, PAI-1 promoted the recruitment and activation of neutrophils in alveolar, thus increasing the anti-infectious effect, but excessive aggregation of neutrophils could exacerbate the injury of alveolar in the following period. It is of great importance to take the advantage of PAI-1 to maximize the anti-infectious effect of neutrophils, and at the same time, minimize the damage in ALI. PAI-1/PECAM/p38MAPK/PI3K pathway is closely related with the recruitment and activation of neutrophils. In order to clarify the mechanism of interations between this pathway and neutrophils in alveolar, find out the appropriate therapeutic protocol of PAI-039, an inhibitor of PAI-1, and provide theoretical basis of the therapy aimed at PAI-1, we would carried out this study by LPS-ALI model in PAI-1 knockout mice.

在急性呼吸窘迫综合征（ARDS）/急性肺损伤（ALI）的发生过程中，中性粒细胞起核心作用。凝血纤溶系统对中性粒细胞募集和活化具有重要的调控作用，但机制未明。纤溶酶原激活物抑制物-1（PAI-1）与肺损伤的预后密切相关。在肺损伤早期，PAI-1促进中性粒细胞肺内募集活化，发挥抗感染作用，但晚期中性粒细胞的过度募集活化会加重肺损伤。通过调控PAI-1使中性粒细胞发挥最大抗感染作用，同时又不造成过度肺损伤有重要的治疗意义。PAI-1/PECAM/p38MAPK/PI3K 通路与中性粒细胞的募集活化相关，本研究拟利用PAI-1敲除小鼠建立LPS-肺损伤模型，研究调控该通路对小鼠肺损伤和中性粒细胞肺内募集活化的影响，初步阐明PAI-1在肺损伤中调控中性粒细胞肺内募集和活化的机制，并探索PAI-1抑制剂在肺损伤中的治疗作用以及合适的治疗时间，最终为以PAI-1为靶点的肺损伤治疗提供依据。

急性呼吸窘迫综合征（ARDS）/急性肺损伤（ALI）死亡率高，中性粒细胞在其中起关键作用，但是中性粒细胞在肺内募集活化的机制仍不明确。“免疫性血栓形成”概念的提出揭示了凝血纤溶系统可以通过炎症反应影响ARDS/ALI的预后。本课题拟研究纤溶酶原激活物抑制物-1（PAI-1）在ARDS/ALI中调控中性粒细胞肺内募集活化的机制。我们在大鼠II型肺泡上皮细胞（AECIIs）、C57BL/6野生型和PAI-1敲除（KO）小鼠中采用LPS进行造模，探索（1）PAI-1对LPS介导的ALI的调节；（2）PAI-1调控中性粒细胞募集活化的机制；（3）PAI-1对中性粒细胞胞外诱捕网（NETs）的调控；和（4）ARDS患者出凝血功能与预后的关系。结果表明，LPS使AECIIs活力降低，PAI-1A（等效于大鼠的PAI-1）表达增加，且能释放至胞外。PAI-039抑制AECIIs表达PAI-1A，且能逆转LPS对AECIIs增殖的抑制。小鼠实验表明，LPS气管内滴注能造成肺、支气管肺泡灌洗液（BALF）和血浆中PAI-1增加，滴入致死量的LPS后，PAI-039能加速小鼠死亡。Transwell法进行中性粒细胞和AECIIs培养的结果表明，PAI-1A重组蛋白可减少中性粒细胞死亡，PAI-1A重组蛋白或LPS均可增加中性粒细胞募集和AECIIs死亡，该过程可被PAI-039抑制。PAI-1A重组蛋白可使中性粒细胞相关炎症因子明显上升，以IL-6最明显，而PAI-039显著降低了IL-6表达。LPS损伤AECIIs后，STAT3的Tyr705位点磷酸化显著升高，该过程能被PAI-039抑制。JAK抑制剂AZD1480能减少STAT3的磷酸化，显著减少了AECIIs损伤，我们推测PAI-1通过IL-6-JAK-STAT3通路产生AECIIs损伤。小鼠实验表明，抑制PAI-1能使得肺损伤小鼠BALF中cfDNA/NETs水平降低，与野生型小鼠相比，PAI-1 KO小鼠肺内的NETs明显减少。最后我们回顾性分析了ARDS患者出凝血功能与预后的关系，结果提示PAI-1和D-二聚体与预后相关，PAI-1联合D-二聚体具有较高的敏感性和特异性。综上，本课题揭示了PAI-1在ARDS/ALI中所起的关键作用及其调控中性粒细胞募集活化的机制，为将来相关药物研发提供了理论支持。