PLD2调控mTOR/HIF-1α/VEGF信号通路在脓毒症NETs损害肠黏膜屏障的作用机制

Neutrophil extracellular traps (NETs) produced by neutrophils during sepsis could jeopardize the intestinal mucosal barrier function. However, the details of modulatory mechanism of the production of NETs are still unclear. Our previous preliminary researches demonstrated that mTOR/HIF-1α/VEGF pathway might be involved in the modulatory of NETs production and the upstream modulatory molecule: PLD2 suppressed the expression of VEGF. Based on these data and previous studies, we proposed the possible mechanism of NETs production: During the early stage of sepsis, inflammatory factors elucidated by PMN suppress the expression of PLD2 and thus upregulated the mTOR/HIF-1α/VEGF pathway and promote the production of NETs, which increased the antimicrobial ability and was good for the host; During the late stage of sepsis, PMN was over-activated and the increased PLD2 downregulated mTOR/HIF-1α/VEGF pathway and decreased the production of NETs, which decreased the antimicrobial ability. Histone, produced by NETs degeneration, accumulated at the microcirculation and jeopardized the intestinal mucosal barrier function, thus was bad for the host. In this proposal, using PLD2-KO cecal ligation puncture (CLP) animal model and cell culture experiment, we applied different techniques, like siRNA silence, immunofluorescence, HPLC and PCR. This study may provide the new therapy target and theoretical basis how intestinal dysfunction developed during sepsis.

脓毒症时，肠黏膜屏障功能损害是导致多器官功能障碍发生的最主要原因之一。中性粒细胞活化后产生的胞外陷阱网（NETs）可损害肠黏膜屏障，如何准确调控NETs的合成尚不清楚。前期预实验表明mTOR/HIF-1α/VEGF信号通路参与调控NETs的合成，上游信号转导蛋白PLD2抑制HIF-1α表达。因此我们提出假设：脓毒症早期，PLD2逆向上调mTOR/HIF-1α/VEGF信号通路，促进NETs合成；脓毒症后期，PLD2表达增加后抑制该信号通路，机体杀菌能力下降，NETs主要成分之一组蛋白反而损伤肠黏膜上皮细胞、加重肠黏膜屏障功能障碍。本项目利用PLD2基因敲除模型，通过siRNA基因沉默、免疫荧光、HPLC等技术，阐明脓毒症不同时期PLD2介导的mTOR/HIF-1α/VEGF信号通路调控NETs损伤肠黏膜屏障的分子机制，可为脓毒症肠黏膜屏障功能障碍的发生机制提供新的理论基础和潜在的治疗靶点。

脓毒症时，肠黏膜屏障功能损害是导致多器官功能障碍发生的最主要原因之一。中性粒细胞活化后产生的胞外陷阱网（NETs）能参与肠黏膜屏障功能受损的病理生理过程。 NETs对肠屏障功能的损伤机制尚不清楚。.第一部分研究了NETs在脓毒症时参与对肠屏障功能的损伤。经伦理委员会审核后，课题组收集腹腔脓毒症患者的血清及术中肠道标本，并与对照组（造口还纳组）对比，发现升高的血清NETs及肠道组织中浸润的NETs标志物，与腹部脓毒症患者的肠道损伤有关。利用盲肠结扎穿孔（CLP）技术建腹腔感染的脓毒症小鼠模型，证明PAD4敲除阻断NETs生成，可以减轻脓毒症时肠屏障损伤。通过提取人中性粒细胞（PMN）生成的NETs，体外与Caco2构建的肠单层上皮细胞模型共培养，证明了NETs呈剂量依赖性地损害 Caco2 上皮单层屏障。.第二部分研究了PLD2通过mTOR/HIF-1α通路介导NETs对脓毒症时肠屏障功能。在临床标本中发现脓毒症患者肠道中PLD2、mTOR、HIF-1α的表达水平升高。通过引进PLD2基因敲除鼠并建立CLP脓毒症小鼠模型，表明PLD2敲除可减轻脓毒症时肠屏障的损伤。通过使用PLD2的抑制剂、mTOR的抑制剂及激动剂干预NETs损伤Caco2上皮单层屏障，证明PLD2通过mTOR/HIF-1α途径介导NETs对肠屏障的损伤。.第三部分研究了NETs激活TLR9-ER stress-ROS信号通路介导肠屏障功能损伤。结果表明脓毒症患者肠道中NETs浸润增加，并与 ER Stress激活和肠道凋亡相关。利用腹腔注射脂多糖（LPS）建立脓毒症小鼠模型，证明PAD4 缺乏可改善肠道屏障损伤并减轻感染性休克中的肠道炎症和 ER Stress激活。在动物实验及细胞实验中，发现NETs激活ER Stress，ER Stress的抑制减轻了NETs对肠上皮细胞的损伤。应用TLR9拮抗剂发现，NETs诱导的ER Stress激活受到抑制，从而减轻肠上皮细胞损伤。.本研究为脓毒症肠黏膜屏障功能障碍的发生机制提供新的理论基础和潜在的治疗靶点。