WISPI-整合素通路在机械通气诱导的肺损伤中的机制研究

CCN proteins triger cell signalng pathway by binding integrin receptor. Separately it has found that integrin signaling pathway activation plays a key role in acute lung injury.Using Genome-wide Association Study (GWAS) with 23 inbred mouse strains in lung injury model, we identified that WNT1 inducible signaling pathway protein 1 (Wisp1) gene was one of the significant single nucleotide polymorphisms. We demonstrated the synergistic effect of WISP1 and TLRs agonists on inflammatory cytokine secretion in macrophages. The current study is to uncover the mechanism of WISP1 in the synergistic effect associated with TLRs signalng transduction. Our hypothesis is that activation of TLRs sigaling pathway would consequently upregulate integrins gene expression in both mRNA and protein level which results in wisp1 signaling by binding its receptors-integrins. Thus,synergistically enhances TLRs singaling pathway. On the other hand, Wisp1-integrins ligation would affect TLRs receptor assembling and strength its signaling.In this study, we will firstly verify the association of TLRs activation and expresion of integrins. Study the upregulation of integrins expression and wisp1 signaling activation in cell biology and molecular biology levels.Conversely,we will investigate whether WISP1-integin ligation would affect TLRs assembling on the lipid raft and strength its signal transduction. Further,we will dissect the signal pathways involved in the synergistic effect of wisp1 and TLRs agonists.Finally, we will verify these findings in VILI animal model by blocking integrin pathways. These studies will provide scientific basis for potential novel proventive therapy for acute lung injury.

CCN蛋白能通过结合整合素受体激活信号通路，而整合素信号通路的激活在急性肺损伤中发挥重要作用。我们前期通过全基因组关联分析发现WISP1是机械通气诱导肺损伤的易感基因，并且在巨噬细胞实验中发现WISP1能通过增强LPS、polyI：C作用，显著放大炎症反应。本研究拟揭示WISP1与TLRs激动剂协同作用机制。首先研究WISP1与LPS、polyI:C分别和共同刺激巨噬细胞后膜表面整合素受体表达及分布的变化，以及对TLR4受体复合物向脂筏募集过程的影响，明确整合素与TLR4信号通路的关系；其次探讨WISP1-整合素通路激活是否与LPS信号通路存在信号增强效应；最后通过体内实验敏感鼠和耐受鼠VILI模型，明确干预整合素信号通路对肺功能的影响，以验证WISP1在VILI中的作用机理，为急性肺损伤的防御及治疗提供新的理论。

背景：前期研究发现WISP1介导机械通气肺损伤，并且在巨噬细胞中与LPS协同性增加促炎细胞因子TNF-α分泌。WISP1通过与细胞膜整合素受体结合后，激活信号通路而具有多种调控功能。本研究在细胞与动物模型中探讨了WISP1-整合素信号通路在炎症发生中的作用机制。.方法：在细胞水平1)分离野生型原代腹腔巨噬细胞，观察WISP1与TLRs激动剂共同刺激后，对细胞分泌TNF-α的影响。2)阻断整合素通路或分离整合素β3基因敲除小鼠原代细胞，给予WISP1与TLRs激动剂共同刺激，观察阻断整合素通路对促炎细胞因子的影响。3)通过LPS或PolyI:C激活相应TLR通路后，观察整合素αvβ3的表达。4)通过分离TLR通路相关基因敲除小鼠原代细胞，给予TLR激动剂刺激，观察整合素αvβ3的表达，以及WISP1与TLRs激动剂共同刺激对TNF-α分泌的影响。在动物水平1）检测机械通气复合Poly:IC诱导急性肺损伤模型肺组织WISP1、整合素β3的表达，在体验证TLR通路对整合素表达的影响2）观察阻断WISP1-整合素通路后对小鼠急性肺损伤的影响。.结果：1）WISP1与TLRs激动剂共同刺激腹腔巨噬细胞后，协同性增加TNF-α分泌。2）阻断整合素β3通路或在整合素β3-/-的细胞中， WISP1与TLRs激动剂协同增加TNF-α分泌的作用消失。3) 分离野生型原代巨噬细胞给予TLRs激动剂刺激，上调整合素αvβ3的表达。4)在TLR相关基因敲除的原代细胞中TLRs激动剂上调整合素表达作用显著性降低，WISP1与TLRs激动剂协同增加TNF-α分泌的作用减弱。5) 在动物模型中，我们发现机械通气复合Poly:IC后，肺组织中WISP1、整合素β3表达上调，免疫共沉淀检测发现WISP1与整合素β3存在相互作用。6) 给予WISP1抗体阻断WISP1-整合素通路后，肺损伤明显减轻。.结论：WISP1通过整合素αvβ3受体，增强TLRs激动剂刺激引起的炎症反应强度，放大炎症反应。而TLRs信号通路的激活反过来又增强整合素的表达，形成正反馈，增强WISP1的协同促炎作用。在机械通气复合Poly：IC模型中发现WISP1与整合素β3存在相互作用，阻断WISP1-整合素通路后，可以明显减轻小鼠肺损伤。我们认为这种正反馈弧的形成可能是WISP1-整合素和TLR通路协同促炎的机理。