5-羟色胺通过调节中性粒细胞NETosis参与脓毒症发展的机制研究

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It is a syndrome of physiologic, pathologic, and biochemical abnormalities induced by infection, which is a major public health concern and common disease in clinic. Neutrophils (polymorphonuclear, PMN) are crucial components of the innate immune response in the process of sepsis, which can eliminate invading microorganisms by secreting antimicrobial proteins as well as by phagocytic uptake and intracellular killing. In addition, it has recently been described that activated neutrophils can expel nuclear DNA to form web-like structures containing granular proteins, which are referred to NETosis and characterized by formation of neutrophil extracellular traps (NETs). On one hand, NETs have been shown to exhibit antimicrobial functions by trapping and killing extracellular pathogens in the blood and tissues during infections. On the other hand, NET-associated proteases and granular proteins have been shown to damage epithelial and endothelial cells, disturb microvascular perfusion in the liver and cause hepatic tissue damage in endotoxemia. Activated platelet can aggravate sepsis by inducing NETs formation, but the detailed mechanism is unclear. Based on our previous studies, we found serotonin (5-HT) released by activated platelet can exacerbate sepsis, and the level of 5-HT is associated with NETs content. So, we hypothesize that 5-HT may promote sepsis development by modulating NETosis of neutrophils. We set up sepsis models in TPH1 knockout mice (deficiency of peripheral 5-HT) and wild-type C57BL/6 mice by cecal ligation and puncture (CLP) and explore the effect of 5-HT on sepsis and NETosis; we also induce NETosis in neutrophils and study the effect of 5-HT on NETs formation. Then we study the detailed mechanisms by up-regulate and down-regulate the key 5-HT receptor and signal pathways in vivo and in vitro. The present study can provide a new insights in the mechanism of sepsis development and a new therapeutic target for sepsis.

脓毒症是临床上常见的危重症疾病，中性粒细胞（PMN）作为机体固有免疫系统的重要组成部分在该疾病中发挥关键作用。新近研究发现PMN的特殊死亡方式—NETosis可以促进脓毒症的进展，而血小板激活是其中关键步骤，然而具体机制尚不清楚。我们前期发现，血小板激活后释放的5-羟色胺（5-HT）能够加重脓毒症，其释放水平与中性粒细胞胞外诱捕网（NETs）水平密切相关。因此，我们猜想5-HT可能通过调节NETosis及相关通路参与脓毒症的发展。本研究拟通过TPH1敲除小鼠（外周型5-HT缺失）和野生型小鼠的脓毒症模型以及利用PMN建立NETosis体外模型后给予5-HT干预，研究NETs形成及相关机制；分别通过上调及下调5-HT受体和相关信号通路后观察小鼠脓毒症的情况，NETs的形成和功能，明确5-HT、NETs及脓毒症之间的关系。本项目将为脓毒症的发病机制奠定新的理论基础，为寻找新的治疗方案提供思路。

脓毒症是临床上常见的危重症疾病，中性粒细胞（PMN）作为机体固有免疫系统的重要组成部分在该疾病中发挥关键作用。新近研究发现PMN的特殊死亡方式—NETosis可以促进脓毒症的进展，而血小板激活是其中关键步骤，然而具体机制尚不清楚。血小板激活后释放的5-羟色胺（5-HT）能够加重脓毒症，其释放水平与中性粒细胞胞外诱捕网（NETs）水平密切相关。因此，我们在本研究中主要研究了5-HT、NETosis及脓毒症的关系。首先，在临床资料及血液标本层面发现，脓毒症患者中5-HT，NETosis相关指标cf-DNA/NETs及MPO-DNA水平均显著高于健康志愿者；随着脓毒症的严重程度从轻到重，血清中5-HT，cf-DNA/NETs及MPO-DNA水平均显著增高；且5-HT与cf-DNA/NETs和MPO-DNA进水平均呈显著性正相关。从动物实验层面发现，当小鼠体内富含5-HT（野生型小鼠、TPH1敲除小鼠+5-HTP、野生型小鼠+氯吡格雷、野生型小鼠+ADP）时，通过CLP造模后其脓毒症的临床评分、各脏器组织的病理损伤、炎症反应、组织细菌负荷等指标均显著增高；而当小鼠体内缺乏5-HT（TPH1敲除小鼠）时，其脓毒症损伤明显减轻。在细胞实验层面，通过给予甲氧基苯丙胺（PMA）和脂多糖（LPS）诱导HL-60 细胞系NETosis 的产生，发现5-HT的干预能够影响NETs相关蛋白如Cit-H3,Ly6G的表达，而这一途径是通过影响5-HT7/Gα12/mTOR信号通路来实现的。本课题的开展深入探讨 5-HT调控脓毒症发展的新机制，并为寻找新的脓毒症治疗策略提供有利条件。