THROMBIN AND INFLAMMATORY TISSUE INJURY

革兰氏阴性菌感染引起的脓毒症是一个主要的临床问题，与许多病理生理改变有关，包括肝功能衰竭。脓毒症时对肝脏的损害是由细菌脂多糖(LPS)介导的。内毒素诱导的肝损伤需要PAR-1凝血酶受体的激活，凝血酶的这种作用可能会刺激趋化因子的释放，促进中性粒细胞(PMN)从肝窦到肝实质的跨内皮迁移。在其他系统中，凝血酶促进中性粒细胞的跨内皮细胞迁移并刺激趋化因子的释放(即。CINC-1)是PMN迁移所必需的。因此，该方案中的研究将验证以下假设：在内毒素诱导的肝脏损伤过程中，凝血酶刺激趋化因子的释放，这是PMN从肝窦向实质迁移所必需的，并且凝血酶的这一作用是通过PAR-1受体介导的。研究将首先利用免疫组织化学方法和/或电子显微镜确定肝脏中哪些细胞类型表达凝血酶受体。然后，将使用包括免疫组织化学在内的形态学技术来确定抑制凝血酶是否会在体内和分离的灌流肝脏中减少大鼠暴露于脂多糖后PMN的跨内皮迁移。我们将使用蛋白质印迹分析、酶联免疫吸附试验和定量聚合酶链式反应来确定在脂多糖诱导的肝损伤过程中，是否需要凝血酶来诱导和释放趋化蛋白CINC-1。使用肝细胞原代培养的研究将确定凝血酶诱导的PMN迁移和CINC-1释放所需的肝细胞类型。这些研究的结果将阐明凝血酶在炎性肝损伤中发挥关键作用的机制，并可能为脓毒症的临床干预提供有用的策略。

Sepsis resulting from gram-negative bacterial infections is a major clinical problem associated with many pathophysiological alterations, including liver failure. Damage to the liver during sepsis is mediated by bacterial lipopolysaccharide (LPS). Activation of the PAR-1 thrombin receptor is required for LPS-induced liver injury, and this action of thrombin may stimulate the release of a chemotactic factor that promotes transendothelial migration of neutrophils (PMNs) from the liver sinusoids into the hepatic parenchyma. In other systems, thrombin promotes transendothelial migration of PMNs and stimulates the release of chemotactic factors (ie. CINC-1) necessary for PMN transmigration. Therefore, the studies in this proposal will test the hypothesis that thrombin stimulates the release of a chemotactic factor that is necessary for transmigration of PMNs from the sinusoid into the parenchyma during LPS-induced liver in injury and that this action of thrombin is mediated through the PAR-1 receptor. Studies will first determine which cell types in the liver express the thrombin receptor using immunohistochemical methods and/or electron microscopy. Morphologic techniques, including immunohistochemistry, will then be used to determine if inhibition of thrombin attenuates transendothelial migration of PMNs after exposure of rats to LPS in vivo and in the isolated, perfused liver. Western blot analysis, ELISA and quantitative PCR will be used to determine if thrombin is required for the induction and release of the chemotactic protein, CINC-1, during LPS-induced liver injury. Studies using primary cultures of liver cells will determine which liver cell types are required for thrombin-induced PMN transmigration and CINC-1 release. Results of these studies will elucidate the mechanism by which thrombin exerts its critical action during inflammatory liver injury and may lead to a useful strategy for clinical intervention during sepsis.