Btk在创伤后失血性休克诱发的全身炎症反应和肺损伤中的作用和机制

Bruton's tyrosine kinase (Btk) belongs to the Tec kinase family s of nonreceptor tyrosine kinases. Recent studies have shown that Btk plays an important role in the signaling transduction of Toll-like receptor 4 (TLR4), but the importance of Btk in the regulation of immune and inflammatory responses in vivo remains largely unknown. Since TLR4 signaling pathway is crucial for the pathogenesis of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS)following severe trauma, we hypothesized that Btk, the key signaling molecule in the TLR4 signal pathway, may also have a important role in mediating the local and systemic inflammatory response and multiple organ injury in the course of traumatic shock. In this study, we investigated the role and the underlying molecular mechanisms of Btk in the development of systemic inflammatory response and acute lung injury in a mouse model of traumatic shock caused by hemorrhagic shock with bilateral femur fracture. We examined the expression and activation of Btk in multiple organs including the lung. We also determined the role of Btk signal in lipopolysaccharide-induced expression of proinflammatory cytokines and the activation of MAPKs and NF-kB signaling pathways by peritoneal macrophages as well as by alveolar macrophages. We further studied the in vivo significance of Btk signal in the pathogenesis of systemic inflammatory response and acute lung injury through specific blockade of Btk expression by means of adoptive transfer of peritoneal macrophages and alveolar macrophages having been infected with a lentiviral vector harboring RNAi sequence targeting the mouse Btk gene. This study will help reveal a key pathophysiological role of Btk in the development of systemic inflammatory response and multiple organ injury following severe trauma and might thus allow us to consider Btk as a therapeutic target against the trauma-induced SIRS and MODS.

Bruton酪氨酸蛋白激酶（Btk）是非受体酪氨酸蛋白激酶Tec家族成员之一，近年研究发现它是TLRs信号通路的一个重要信号分子，但它在体内炎症反应中的作用仍知之甚少。由于TLRs信号通路在创伤后SIRS和MODS的发生发展过程中具有重要作用，我们推测Btk信号可能通过激活下游促炎性信号通路，参与创伤后全身炎症反应和重要脏器损伤的发病过程。为此，本项目拟以临床常见的多发性骨折合并失血性休克为致伤因素，复制小鼠创伤性休克模型，观察Btk激酶在各脏器中的表达及活化，并以腹腔巨噬细胞和肺泡巨噬细胞为研究对象，采用慢病毒载体RNA干扰方法研究Btk在内毒素诱导的MAPK和NF-kB活化及细胞因子表达中的作用，并采用过继巨噬细胞治疗实验研究Btk在创性休克诱发的全身炎症反应和肺损伤中的作用和机制。本项目将有助于阐明严重创伤后失控性炎症反应的发生机制，为创伤后MODS的防治提供新的思路。

Bruton酪氨酸蛋白激酶（BTK）是酪氨酸蛋白激酶Tec家族成员之一，在B细胞发育以及成熟的B细胞活化和生存中起到了至关重要的作用。已有研究表明BTK的活化可能刺激NF-κB和MAPK信号通路并最终引发一系列炎症反应。BTK抑制剂可能在控制B细胞恶化和B细胞相关的自身免疫性疾病中具有疗效；然而BTK是否参与了THS诱导的肺损伤仍有待阐明。本研究旨在探讨BTK在THS诱导的肺损伤过程中的作用并检测BTK抑制剂对肺部的保护作用。雄性SD大鼠被随机分成4组：i) 假手术组；ii) THS诱导损伤组；iii) 创伤前接受BTK抑制剂LFM-A13治疗组；iv) 无创伤手术前接受BTK抑制剂LFM-A13治疗组。Western Blot和免疫组化法检测磷酸化BTK的表达。考马斯亮蓝染色法检测肺泡灌洗液中的总蛋白浓度，吉姆萨染色法检测肺泡灌洗液中白细胞和嗜酸性粒细胞的数量，ELisa法检测外周血炎症因子。HE染色观察肺组织形态学改变。肺组织NO含量和iNOS表达量通过NO试剂盒和western blot技术进行分析。NF-κB和MAPK信号通路的活化通过western blot技术和EMSA技术进行检测。结果表明BTK在THS组大鼠肺组织被激活。THS组大鼠肺泡灌洗液中总蛋白浓度，白细胞和嗜酸性粒细胞数量，外周血中IL- 1β、IL- 6 、TNF-α、MCP- 1因子表达量显著上升，经LFM-A13治疗后各项指标显著下降。THS诱导产生的间质增生，肺水肿和中性粒细胞浸润现象可通过BTK抑制剂改善。此外，NO的释放，iNOS的过表达以及NF-κB和MAPK信号通路通过BTK抑制剂所抑制。表明BTK可能在THS诱导的肺损伤的发病机理中具有关键的作用，并且BTK抑制剂可以明显减轻THS诱导的肺损伤。这些结果为THS诱导的肺损伤的治疗提供了潜在的依据。