HSF1对脓毒症时血小板-白细胞-内皮细胞相互作用及多器官功能障碍的影响

In sepsis, the activation and interaction of platelets, leukocytes and endothelial cells result in coagulopathy and multiple organ dysfunction syndrome (MODS). So far, it is not clear yet that what effect of heat shock factor 1(HSF1) has on the activation and interactions of platelets, leukocytes and endothelial cells in sepsis. In recent years, the applicant found in septic mice that knock out of HSF1 gene resulted in exacerbation of multiple organ damage, shortening of bleeding time, increase in activation and interaction of platelets, leukocytes and endothelial cells, and identified intact HSF1 binding sites in the promoter regions of multiple genes of adhesion molecules, chemokines and their receptors, and coagulation related factors. Therefore, it was hypothesized that during sepsis HSF1 could inhibit the activation and interactions of platelets, leukocytes and endothelial cells, alleviate coagulopathy and the changes in permeability of micro-vessels, and relieve MODS by regulating directly the expression of multiple genes. In this project we plan to prepare a septic model using HSF1 knockout mice, investigating the effect of HSF1 on the activation and interaction of platelets, leukocytes and endothelial cells, coagulopathy and MODS and the underlying mechanism from intact animal, cellular and molecular levels, and clarifying the direct transcriptional regulation of HSF1 for related target genes by using a set of technologies in gene expression regulation. This project is characterized by its original innovation. It is expected that this project will elucidate the cellular and molecular mechanisms by which HSF1 alleviates MODS in sepsis from a completely new angle of view, and provide new targets and experimental evidences for the intervention of sepsis.

脓毒症时血小板(P)、白细胞(L)、血管内皮细胞(E)被激活并发生相互作用，导致凝血功能紊乱和MODS。HSF1对脓毒症时P、L、E的活化及其相互作用有何影响目前仍不清楚。申请者近年发现，HSF1基因敲除导致脓毒症小鼠器官损伤加重，出血时间缩短，P、L、E活化及三者相互作用增加，且发现多种粘附分子、趋化因子及其受体、凝血相关因子启动子区含HSF1的结合位点，故提出假设：HSF1可直接调控多种基因表达而抑制脓毒症时P、L、E的活化及其相互作用，改善凝血功能紊乱和微血管通透性变化，减轻MODS。本项目拟采用HSF1基因敲除小鼠制备脓毒症模型，从整体、细胞及分子水平揭示HSF1对P、L、E活化及其相互作用、凝血功能紊乱和MODS的影响及其机制，并从转录水平阐明HSF1对相关靶基因的直接调控。本项目预期从一全新角度揭示HSF1减轻脓毒症MODS的细胞分子机理，为脓毒症的干预提供新的靶点和实验证据。

我们前期发现，HSF1抑制内毒素血症小鼠多种炎症因子的表达，减轻全身炎症反应，减轻多器官损伤。然而，HSF1是否参与调节脓毒症时凝血功能紊乱，是否影响血小板-白细胞-血管内皮细胞的活化，是否参与凝血相关基因的转录调控，尚未见研究报道。本项目采用HSF1基因敲除小鼠制备脓毒症模型，从整体、细胞及分子水平对上述问题进行了深入探讨，主要发现如下：.1.血小板拮抗剂氯吡格雷可抑制内毒素血症小鼠血小板、白细胞及内皮细胞的激活，并抑制血小板-白细胞-内皮细胞的相互作用。.2.HSF1抑制脓毒症小鼠凝血功能的过度激活，减少微血栓的形成，减轻凝血功能紊乱，从而减轻了脓毒症小鼠的多器官损伤。.3.HSF1抑制脓毒症小鼠血小板的活化，抑制血小板和白细胞的相互作用以及白细胞表面组织因子的表达。.4.HSF1上调脓毒症小鼠血浆、组织中t-PA，tfpi2和protein C等抗凝基因的表达。经HSF1过表达或RNA干扰后，发现HSF1促进LPS所致内皮细胞中t-PA，tfpi2和protein C等抗凝相关基因mRNA和蛋白质的表达上调。.5.采用凝胶电泳迁移实验（EMSA）和双荧光素酶报告基因实验发现HSF1可与t-PA，tfpi2以及protein C基因启动子区的HSE结合，直接启动上述抗凝血相关基因的转录，从而减轻脓毒症时的凝血功能紊乱。.6.HSF1通过直接下调MCP-1/CCR2的转录，抑制巨噬细胞在肺组织的浸润，从而减轻LPS诱导的小鼠急性肺损伤。.上述研究已发表论文12篇，待发表论文5篇。本项目从抗凝血功能障碍的全新角度揭示了HSF1减轻脓毒症多器官损伤的细胞分子机理，为揭示脓毒症的发病机制和寻找新的干预措施提供了重要实验证据。.项目负责人肖献忠教授多次受邀出席国际国内学术会议并作学术报告，作为中国代表参与制订脓毒症临床前研究国际专家共识，并当选国际休克学会联盟理事。