重症中暑HMGB1调控血管内皮TF表达促进血栓形成机制研究

Disseminated intravascular coagulation(DIC) is one of the important pathogenesis in severe heatstroke-induced multiple organ dysfunction syndrome(MODS), while vascular endothelial injury-induced thrombus formation is the key link in the DIC caused by severe heatstroke where tissue factors(TF) is pivotal promoter. The vascular endothelial platform-based inflammation and coagulation disorder are two crucial aspects in severe heatstroke,in which TF is the key target. It is found that many inflammatory factors could regulate TF expression. It is found in our previous studies that High mobility group box 1(HMGB1), a key inflammatory factor, was a outcome-predicting parameter that elevated in early stage and maintained for a long time in heatstroke.It is indicated that HMGB1 could promote TF expression in vascular endothelial cell subjected to heat in vitro and pretreatment with HMGB1 antibody could decrease hepatic sinusoid endothelial damage and thrombus formation in hepatic sinusoid in severe heatstroke rat models in our previous study.It is recently reported that HMGB1 can induce vascular endothelial TF expression. However, whether HMGB1 has a role in TF expression regulation to promote thrombosis and its involved molecular mechanism in severe heatstroke is unproved yet. We hypothesized that early elevated and maintained high level of HMGB1 may regulate the TF expression to accelerate thrombosis to induce DIC and MODS in severe heatstroke. HMGB1 regulating vascular endothelial TF expression which promote thrombus formation in severe heatstroke and its related molecular mechanism are explored in our study.

DIC是重症中暑MODS发生的重要机制，血管内皮损伤诱发血栓形成是启动DIC的关键环节，TF是血栓形成的关键因子。基于血管内皮平台的炎症反应和凝血紊乱网络调控是重症中暑根本环节，TF是其网络调控中的重要靶点，多种炎症介质参与了TF表达调控。HMGB1为重症中暑重要的炎症介质，我们发现HMGB1早期升高且长时间维持，为中暑预后不良指标。同时发现HMGB1可诱导热打击血管内皮TF表达，HMGB1抗体预处理可改善重症中暑大鼠肝窦内皮细胞损害和肝窦血栓形成。近期研究发现HMGB1可调控血管内皮TF表达，重症中暑HMGB1是否参与调控血管内皮TF表达促进血栓形成及其分子机制如何未见研究。基于此我们提出假设：重症中暑HMGB1早期升高可能调控血管内皮TF表达，促进血栓形成诱发DIC发生，参与MODS进展。本项目探讨重症中暑HMGB1调控血管内皮TF表达促进血栓形成分子机制。

本项目基于重症中暑血管内皮细胞损伤炎凝核心发病机制，探讨HMGB1诱导血管内皮细胞血栓形成分子机制。基于大鼠模型和临床患者发现重症中暑存在动态凝血紊乱，低凝状态与病情严重程度显著相关且可预测预后。体外热打击诱发血管内皮细胞促凝表型，表现为TF、vWF和PAI-1分泌温度依赖性升高，t-PA分泌温度依赖性降低。重症中暑大鼠组织病理学表现为微血栓形成、血管周边为著的炎症细胞浸润和细胞变性坏死。基于外泌体新角度探讨重症中暑血管内皮损伤机制，结果发现热打击HUVECs释放外泌体lncRNAs、mRNAs和microRNAs（miRs）存在明显差异，其靶蛋白涉及炎症、凝血、凋亡和心血管功能等。进一步分析发现差异最显著外泌体miR-1-3p对热打击血管内皮细胞发挥促凝和细胞毒性作用，参与血管平滑肌细胞收缩功能障碍。探讨热打击组织血管损伤机制，发现p38和ERK对热打击肺微血管内皮细胞（PMVECs）发挥抗凋亡和通透性保护作用，JNK发挥促凋亡和增加通透性作用。体内外探讨HMGB1诱导热打击血管内皮凝血表型分子变化机制发现：重症中暑患者和大鼠早期血清HMGB1与凝血指标同趋势升高，且与预后显著相关；大鼠热应激前注射HMGB1中和抗体，肝、肺、肾出血和血栓形成和肝窦内皮细胞肿胀缓解；热打击HUVEC诱导HMGB1胞核胞浆转位表达分泌及TLR2受体和核因子Egr-1呈时间依赖性表达升高；凝血因子TF、vWF和t-PA呈时间依赖性升高，TFPI、TM和PAI-1呈时间依赖性降低。拮抗HMGB1和TLR2及抑制Egr-1缓解热打击HMGB1诱导HUVEC分泌凝血因子紊乱。探讨重症中暑预后标记物，发现血浆Ang II和Ang II/Ang-（1-7）可预测重症中暑肝损害发生、血浆外泌体microRNA-1-3p和组蛋白H3明显升高与病情严重程度，可预测预后。探讨重症中暑炎凝反应治疗措施，发现血必净改善重症中暑大鼠微动脉和微静脉红细胞流速、血管管径和血流量、维持平均动脉压、减少体重丢失和改善生存时间；氯沙坦可能通过抑制热打击肝细胞ROS诱导的HMGB1缓解IL-1β和IL-18炎性反应；氢化可的松诱导重症中暑主动脉α1肾上腺素能受体表达改善去甲肾上腺素血管反应性。