“上皮细胞WISP1与巨噬细胞HMGB1促炎环路”在机械通气加重脓毒症肺损伤中的作用与机制

Sepsis induced lung injury often needs mechanical ventilation .Mechanical ventilation synergistic aggravated sepsis induced lung injury but the underline mechanism is still unknown. Our previous studies have shown that moderate tidal volume of mechanical ventilation does not cause lung injury in healthy mice, but increases WISP1 and HMGB1 expression in the lung of septic mice and aggravates secondary injury. In vitro, WISP1/LPS co-stimulation increases HMGB1 release from macrophages, then HMGB1 promotes WISP1 secretion in lung epithelial cell. Therefore, this study focuses on epithelial-macrophage interaction: WISP1-HMGB1 positive signaling pathway in sepsis induced lung injury combined with mechanical ventilation. In the current study: firstly, investigate the expression and distribution of WISP1 in the lung of sepsis combined mechanical ventilation model and the effect of WISP1-integrin pathway inhibition in the lung injury and HMGB1 expression; secondly, investigate the effect of cell specific HMGB1 in sepsis combined mechanical ventilation induced lung injury and the WISP1 expression; thirdly, using LPS/ mechanical stretch co-stimulus model or epithelial cells and macrophages co-culture model to study the mechanism of WISP1-HMGB1 positive feedback pathway in vitro; Lastly, confirm the effect of “WISP1-HMGB1 positive loop” inhibition by intratracheal injection of recombinant protein or neutralization antibody on lung injury in vivo. This project provides a new strategy for the prevention and treatment of sepsis combined with mechanical ventilation induced lung injury.

脓毒症肺损伤常需机械通气支持，但目前脓毒症肺损伤研究中较少考虑机械通气协同损害作用，更不明其机制。前期研究证明：中等潮气量机械通气不会造成健康肺的损伤，但增加脓毒症小鼠肺组织中WISP1和HMGB1分泌，加重脓毒症肺损伤；体外WISP1/LPS刺激巨噬细胞释放HMGB1，HMGB1又反作用于肺上皮细胞促进WISP1分泌。因此本研究聚焦“上皮细胞与巨噬细胞间WISP1-HMGB1环路”在脓毒症机械通气复合肺损伤中的机制：1、在小鼠复合肺损伤模型中检测WISP1表达与分布，及干预 WISP1-整合素通路对肺损伤及HMGB1的表达影响；2、检测细胞特异性HMGB1在复合肺损伤中的作用及对WISP1的表达影响；3、体外采用双细胞共培养或LPS/机械牵拉共刺激模型，明确此正反馈环路的存在；4、经气道给药观察干预该环路的疗效，在体验证在肺损伤中的作用。本研究为理解和防治脓毒症机械通气肺损伤提供新思路。

脓毒症肺损伤常需机械通气支持，但目前脓毒症肺损伤研究中较少考虑机械通气协同损害作用，更不明其机制。前期研究证明：中等潮气量机械通气不会造成健康肺的损伤，但增加脓毒症小鼠肺组织中WISP1和HMGB1分泌，加重脓毒症肺损伤；体外WISP1/LPS刺激巨噬细胞释放HMGB1，HMGB1又反作用于肺上皮细胞促进WISP1分泌。因此本研究采用盲肠结扎穿孔诱导脓毒症复合机械通气的在体模型与巨噬细胞-上皮细胞共培养的体外模型探讨“上皮细胞与巨噬细胞WISP1-HMGB1环路”在脓毒症机械通气复合肺损伤中的机制。.研究结果发现：①脓毒症小鼠复合中等潮气量机械通气，肺损伤明显加重，但是该协同作用在TLR4基因敲除小鼠中消失。气管内给予野生型小鼠WISP1或整合素β5中和抗体可以减轻脓毒症复合机械通气肺损伤。离体巨噬细胞实验表明LPS/WISP1序列刺激协同增强细胞因子和细胞趋化因子的释放，整合素β5 siRNA可以阻断LPS/WISP1协同促炎效应，该协同效应在TLR4基因敲除小鼠巨噬细胞消失；②与C57BL/6小鼠相比，LyzHMGB1和iHMGB1基因敲除小鼠炎症水平、MAPK信号通路的激活程度、中性粒细胞募集、急性肺损伤程度下降；虽然EC HMGB1基因敲除小鼠炎症水平、MAPK信号通路激活程度未降低，但是中性粒细胞募集、急性肺损伤程度下降；③在上皮-巨噬细胞共培养体系中，巨噬细胞显著增加上皮细胞分泌WISP1，依赖巨噬细胞源性HMGB1，上皮细胞WISP1能够显著增加巨噬细胞分泌炎症因子（TNF-α、IL-6）和趋化因子（MCP-1、MIP-2），LPS/WISP1具有协同促炎效应；④气管内给予WISP1抗体可以降低脓毒症小鼠肺泡巨噬细胞分泌炎症因子和趋化因子，降低血浆和肺泡灌洗液中的炎症水平，改善小鼠生存率。.“上皮细胞-巨噬细胞交互效应：WISP1-HMGB1正反馈通路” 补充与完善急性肺损伤内在机制，为预防和治疗肺损伤提供新策略。