肺内皮细胞S1PR1受体在流感病毒所致ARDS中的作用

nfluenza virus-inducing ARDS following the cytokine storm during influenza infection plays a key role in the motality.Pulmonary endothelium were recently recongnised as an important soure of cytokines.Excitment of S1PR1 on endothelium would efficiently reduced the level of cytokine storm.However, how did excitement of S1PR1 reduce the produce of cytokine in endothelum and which kind of pulmonary endothelium were the key source of cytokine? We have demonstrated that mRNA levels of cykokines were increased after human pulmonary microvascular endotheliar cells were infected by influenza virus.Based on those, we speculate that human vascular endothelium were the main source of cytokine storm and excitement of S1PR1 would influence signal pathways responsible for produce of cytokines.To vertify this hypothesis,we would like to generate the S1PR1 silencing/over-expressing human vascular endothelial cell lines respectively in dissection of the influence of signal pathways responsible for produce of cytokines through the excitment of S1PR1. We will then use vascular endothelial S1PR1 gene knockout mice to illustrate the significance of S1PR1 on vascular endothelium in the regulation of cytokine storm during influenza infection. We anticipate that excitment of S1PR1 on vascular endothelium may become a new strategy for the treatment of influenza virus-inducing ARDS.

炎症反应引发的ARDS是流感患者死亡的首要原因，最新研究证实肺内皮细胞是流感所致炎症反应的枢纽环节，激动其S1PR1受体可显著降低炎症程度。目前，参与流感炎症反应的肺内皮细胞类型仍不清楚，且S1PR1受体调控肺内皮细胞促炎因子产生的机制不明。本课题前期研究证实甲型H1N1病毒感染人血管内皮细胞后可诱导促炎因子产生及细胞凋亡，S1PR1特异性激动剂可抑制促炎因子产生。据此我们认为：血管内皮细胞参与流感病毒感染后炎症反应，激动S1PR1受体可影响炎症相关信号通路。本课题拟建立S1PR1受体沉默/高表达人血管内皮细胞株，探究激动S1PR1受体是否通过调控NF-kB、MAPK信号通路影响促炎因子产生；然后使用血管内皮细胞S1PR1基因敲除小鼠，构建流感病毒感染模型，明确血管内皮细胞在流感所致炎症反应中地位。本项目期望通过干预肺内皮细胞S1PR1受体调控炎症反应为临床治疗流感所致ARDS提供新策略。

甲型流感病毒是导致病毒性肺炎和ARDS最常见的病原体之一。急性呼吸窘迫综合征(acute respiratory distress syndrome，ARDS)是最常见的重症急性呼吸衰竭，容易被漏诊，病死率高，到目前为止，治疗手段缺乏。既往认为呼吸道上皮细胞在流感感染炎症风暴中扮演主要角色，因其是流感病毒感染复制的主要场所，并且可分泌多种炎症及趋化因子，募集大量专职免疫细胞浸润肺部，破坏肺微血管内皮屏障，从而介导了肺损伤的发生。近期一些研究结果发现，内皮细胞在流感病毒感染炎症风暴中起主要作用，特异性激动肺内皮细胞表面鞘氨醇-1-磷酸受体1（sphingosine-1-phosphate receptor 1，S1PR1）可显著降低流感病毒感染小鼠肺部促炎因子水平，抑制免疫细胞的肺部浸润。.该项目通过构建鞘氨醇-1-磷酸受体1（S1PR1）受体沉默及高表达的人肺微血管内皮细胞株，给予H1N1流感病毒感染及S1PR1特异性激动剂的刺激，分析了细胞炎症因子、趋化因子和黏附分子的表达水平；同时构建了选择性血管内皮细胞S1PR1基因敲除小鼠模型，复制H1N1流感病毒感染模型，在感染后给予S1PR1特异性激动剂处理，观察小鼠急性肺损伤情况，评判肺部炎症水平。.主要的研究结果如下：H1N1流感病毒感染人肺微血管内皮细胞产生大量炎症因子。激动S1PR1减低流感病毒感染时促炎因子产生。激动S1PR1通过β-arrestin2抑制流感病毒诱导NF-kB活化来减低ICAM-1的表达。动物模型中，敲除S1PR1可以明显加重流感病毒感染导致的免疫介导的肺部损伤程度，S1PR1激动剂治疗可以减轻肺损伤，减少促炎因子的产生。S1P1信号通路的活化并不影响病毒复制，改变病毒载量。激动S1PR1通过介导MAPK和NF-kB两条信号通路来调节肺部炎症。S1PR1激动剂与抗病毒药物联合用药可以获得最佳的治疗效果。.该研究立足于临床实际问题，通过体内和体外实验干预血管内皮细胞S1PR1受体表达，建立流感病毒感染模型，并给予S1PR1受体特异性激动剂处理，明确了S1PR1在调控H1N1流感病毒感染引起的炎症反应中的重要作用。针对S1PR1的特异性受体激动剂或许可以作为未来预防和控制甲型流感爆发的候选药物，为临床上流感病毒感染的治疗提供了新思路。