HMGB1激活血小板NLRP3炎性小体对重症中暑血管内皮损伤的调控机制研究

Multiple organ dysfunction is the leading cause of high mortality rate in severe heatstroke, with the essential pathogenesis of microthrombus and inflammatory reaction due to vascular endothelial injury. It is proved that complicated network forms between inflammatory initiation and thrombosis mediated by the vascular endothelium in multiple critical illnesses including severe heatstroke, in which platelet takes key roles. However, it is seldom reported about its mechanisms of the regulatory role of platelet in vascular endothelium injury in heatstroke. It is found that platelet can stimulate NLRP3 inflammasome to release IL-1β through mitochondria ROS activation, which takes roles on regulating vascular endothelium injury in the recent 2013 research report. HMGB1, a typical DMAP molecule, can also act in a similar way to activate NLRP3 inflammasome induced by mitochondria ROS. Platelet mitochondria ROS,initiated by HMGB1, can induce vascualr endothelium injury.Based on the close relationship between mitochondria ROS and severe heatstroke pathogenesis, combined with our previous results that high HMGB1 levels in early stage of severe heatstroke,and specifically inhibiting HMGB1 and mitochondria ROS can prevent the activation of platelet NLRP3 inflammasome and reduce its regulatory effect on vascular endothelium injury.We assumed that platelet can play regulatory roles in vascular endothelium injury through the mitochondria ROS activation of NALP3 inflammasome induced by HMGB1.In the present project,we explore the molecular mechanism of vascular endothelium injury induced by activated platelet based on NLRP3 inflammasom in heatstroke.

基于血管内皮损伤发生的微血栓和炎症反应是重症中暑发病的关键。重症中暑炎症启动与血栓形成之间存在基于血管内皮为媒介的网络关系，血小板在这种网络关系中发挥重要的调控作用。重症中暑血小板以何机制对血管内皮损伤发挥调控作用未见报道。2013年研究发现血小板可基于线粒体ROS激活NLRP3炎性小体促进IL-1β释放的方式诱导血管内皮损伤。HMGB1为一种典型的DAMP分子可诱导线粒体ROS进而激活NLRP3炎性小体。HMGB1诱导血小板线粒体ROS产生可引起血管内皮损伤。基于线粒体ROS与重症中暑发病密切相关，且我们前期发现重症中暑发病早期HMGB1即呈高水平，HMGB1和线粒体ROS特异性抑制可阻止重症中暑血小板NLRP3炎性小体活化并降低血小板诱导的血管内皮损伤。基于此我们提出假设并期探讨其相关机制：重症中暑血小板可基于HMGB1诱导线粒体ROS触发NLRP3炎性小体激活的方式调控血管内皮损伤。

项目基于重症中暑血管内皮损伤炎凝网络发病机制，探讨血小板是否基于HMGB1诱导NLRP3炎性小体机制调控血管内皮损伤。发现麻醉和清醒重症中暑大鼠平均动脉压和体温反应存在差异，清醒中暑大鼠接受总热负荷量和重度热负荷量大于麻醉中暑组，组织病理损害以血管周边更严重，表现为炎症细胞侵润，充血、出血和微血栓形成，细胞变性、坏死等；血管内淤血，局部灶性出血及血管内皮细胞肿胀。重症中暑血清CEC、vWF和TM水平明显升高，APPT、PT、 FIB和血小板计数及全血凝血指标ACT、CR和PF均发生特征性紊乱。热打击HUVEC细胞活力下降和细胞凋亡增加且呈时间依赖性。内质网应激诱导 UPR通路激活和细胞内Ca2+超载与热打击血管内皮细胞凋亡有关，NK-KB活化抑制ROS累积致MAPKs活化降低发挥细胞凋亡保护作用。热打击诱导HUVEC凝血表型分子vWF、PAI-1和TF温度依赖性分泌升高及t-PA温度依赖性分泌减少；诱导炎症介质HMGB1、TNF-α、IL-1β和IL-6温度依赖性分泌升高。发现重症中暑大鼠血小板计数呈时间依赖性下降，血小板导管逐渐扩张、空泡增多、线粒体膜肿胀、嵴模糊、出现自噬小体且自噬蛋白LC3B、ATG7和 P62表达增加，血小板活化蛋白CD62P表达率呈时间依赖性升高。重症中暑大鼠血小板NLRP3和活化Caspase-1表达及NLRP3炎性小体组装分泌IL-1β呈时间依赖性增加；血清HMGB1及其受体TLR2、TLR4和RAGE表达呈时间依赖性升高，HMGB1基于TLR4/RAGE协同作用方式诱导血小板NLRP3炎性小体激活分泌IL-1β，与血小板减少和功能激活有关。重症中暑大鼠血小板ROS水平呈时间依赖性升高，HMGB1-TLR4/RAGE通过ROS诱导NLRP3炎性小体分泌IL-1β。重症中暑大鼠血小板可基于HMGB1-TLR4/RAGE-ROS-NLRP3炎性小体分泌IL-1β诱导HUVEC炎凝表型，凝血表型分子vWF 、TF和 PAI-1分泌增加及t-PA分泌减少，HMGB1、TNF-α、IL-1β和IL-6分泌增加。血必净降低重症中暑血清LPS、TNF-α、IL-1β和IL-6水平，降低肠道iNOS表达，减轻肠道病理损害和细胞凋亡。SOD预处理保护重症中暑病理性血压下降，改善微动静脉红细胞流速、微动脉管径及血流量，降低微动脉血管异常反应，延长生存时间。