汉滩病毒活化TLR4-TRAF6-SFK信号通路致血管内皮细胞通透性升高的机制研究

Hantaviruses cause two human diseases,hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome(HPS) in North and South America.At present,HFRS remains a serious public health problem in China.The course of the disease includes five typical clinical phases:febrile phase,hypotensive phase,oliguric phase,polyuric phase and convalescent phase.All hantavirus-associated syndromes commonly involve increased microvascular permeability.Thus,capillary leakage is the key pathological feature of HFRS in clinical manifestation (such as hemorrhage,hypotension,shock and renal failure). However,the underlying pathophysiological mechanisms leading to this phenomenon are not fully understood,and there are no specific therapeutic drugs for hantavirus infection presently.The paracellular pathway and the transcellular pathway are main channels of solutes leakage extracellularly.They are associated with disorganization of cellular barrier and formation of caveolae and fission, respectively.Src family protein tyrosine kinases (SFKs) induce the phosphorylation of many important proteins [such as vascular endothelial cadherin (VE-cad)、catenins、talin、vinculin、caveolin-1、dynamin-2] in barrier and caveolae structure.Tumour necrosis factor receptor-associated factor-6 (TRAF6) is an adapter protein for Toll-like receptor (TLR)-mediated nuclear factor-κB (NF-κB) signalling pathway activation that induces the production of pro-inflammatory cytokines and it can activate many kinds of kinases.Our previous studies have demonstrated that Hantaan virus （HTNV）infection triggered TLR4-TRAF6 signaling pathway activation in vascular endothelial cells and activated NF-κB,which induced pro-inflammatory cytokines. However,it is still not fully understood that if TRAF6-mediated TLR4 signal could activate Src family protein tyrosine kinases (SFKs) and increase permeability in vascular endothelial cells.For this reason,an in vitro model of HTNV-infected vascular endothelial cells is established in this study.The SFKs are selected as a breakthrough.Based on principle of signal transduction,some methods,such as siRNA,fluorescent quantitative PCR (FQ-PCR),immunoprecipitation (IP),western blotting (WB),immunofluorescence and permeability assay were used in the study.The goals of this study are to clarify the mechanisms of increased permeability induced by HTNV infection and provide new data for pathogenesis of hemorrhagic fever with renal syndrome (HFRS),therapeutic strategy and drug design.This will provide us with the necessary basis for development of effective therapeutics to prevent or diminish this severe disease.

汉坦病毒引起肾综合征出血热(HFRS)，血管渗漏是其临床表现（出血、低血压休克等）的病理基础，其机制尚不明确，也无特效治疗药物。溶质渗出主要经细胞旁和穿细胞途径，分别与细胞屏障结构受损和质膜小凹的形成与分离有关，而SFK可使其中的关键蛋白（如VE-cad、小凹蛋白-1）磷酸化，破坏屏障结构和增加小凹形成与分离。TRAF6是介导TLR活化NF-κB的接头蛋白，可激活多种激酶。前期研究证实，汉滩病毒可触发血管内皮细胞TLR4-TRAF6信号转导，活化NF-κB。但它能否激活SFK并增加细胞通透性尚不清楚。为此，本课题拟以汉滩病毒感染的血管内皮细胞为模型，以SFK为切入点，采用siRNA、定量PCR、免疫荧光、蛋白印迹和通透性测定等技术，探讨汉滩病毒对TLR4-TRAF6-SFK通路的活化及其对蛋白磷酸化的影响，阐明汉滩病毒致细胞通透性升高的机制，为HFRS发病机制研究和治疗药物设计提供新资料。

汉滩病毒（HTNV）归属于布尼亚病毒科汉坦病毒属，主要感染人血管内皮细胞，引起肾综合征出血热（HFRS）。毛细血管通透性升高是其基本病理特征，但机制尚未完全阐明。本课题以 HTNV 感染的血管内皮细胞为模型，以Src家族蛋白酪氨酸激酶（SFK）为切入点，根据细胞信号转导的原理，采用siRNA、定量PCR、免疫荧光、蛋白印迹（WB）等技术，探讨HTNV对TLR4-TRAF6-SFK信号通路的活化及其对细胞屏障结构蛋白的影响，阐明HTNV感染引起血管内皮细胞通透性升高的机制。结果发现，HTNV感染血管内皮细胞后，TRAF6的表达随着时间出现上升的趋势，而抑制TLR4，再用HTNV感染，TRAF6的表达基本不变。同时发现，YES和SRC分别在HTNV感染 24h和48h后表达显著上升，而LYN和FYN在病毒感染48h后没有变化，SFK的磷酸化水平在病毒感染24h出现上升趋势，提示HTNV感染内皮细胞后可激活YES和SRC。利用siRNA技术下调TRAF6的表达，发现感染 HTNV后SRC 的磷酸化水平显著地下降，随之利用WB检测干扰 TRAF6或抑制SFK后细胞粘附连接蛋白VE-Cad、β-连环蛋白、δ-连环蛋白的表达，发现HTNV 感染后，这3种细胞连接蛋白磷酸化表达均有显著地提高，而抑制TRAF6和SRC后，其磷酸化水平均有下降，提示HTNV可通过TLR4-TRAF6通路激活SRC后影响细胞连接蛋白的表达。最后，利用免疫荧光技术检测发现HTNV感染后VE-Cad的分布发生变化，相邻细胞间的界限变模糊，提示HTNV感染会影响细胞间连接蛋白的表达。. 本研究阐明HTNV通过TLR4-TRAF6通路激活SFK，并磷酸化下游细胞连接蛋白，引起细胞通透性升高，不仅为HFRS发病机制研究增添新了新资料，而且还可针对信号通路进行分子药靶的新药设计，为HFRS治疗药物的研发提供新思路。