肺泡上皮细胞来源的外泌体介导炎症信息的传递在脓毒症肺损伤中的作用及机制研究

Studies have shown that exosomes are closely related to inflammatory response of alveolar macrophages (AM), but there is no report about the role of alveolar epithelial cell (AEC)-derived exosomes in sepsis-induced lung injury. In our initial study we found AEC-derived exosomes in alveolar lavage of patients with the sepsis-induced lung injury, and that the LPS-induced exosomes from AEC can cause AM inflammatory response. With the exploratory application of high-throughput miRNA sequencing, we found there were a large number of miRNAs with differential expression levels in the LPS-induced exosomes from AEC. Therefore, we hypothesize that LPS-induced exosomes mediate inflammatory signal transduction between AEC and AM by transferring miRNA to AM, which leads to the sepsis-induced lung injury. In the proposed project we plan to understand the effect of reducing AM inflammation response and the protective effect on the sepsis-induced lung injury by in vivo/vitro inhibition of LPS to control the generation of exosomes from AEC and to prioritize enhancing-inflammation miRNA in LPS-induced exosomes from AEC by using high throughput gene sequencing and RT-qPCR validation. With this study we anticipate to illustrate the way of inflammatory signal transduction between AEC and AM mediated by LPS-induced exosomes from AEC as well as to explore the mechanisms of the sepsis-induced lung injury caused by exosomes.

研究表明外泌体与肺泡巨噬细胞（AM）炎症反应密切相关，但肺泡上皮细胞（AEC）来源的外泌体在脓毒症肺损伤中的作用未见报道。我们前期在脓毒症肺损伤患者的肺泡灌洗液中发现了AEC来源的外泌体，且LPS诱导的AEC外泌体可以引起AM炎症反应，我们探索性地对LPS诱导的AEC外泌体miRNAs进行高通量测序，发现其内含有多种炎症相关miRNAs。故我们假设AEC外泌体通过向AM传递miRNAs，介导了AEC与AM间炎症信息的传递，是脓毒症肺损伤的重要环节。本项目拟观察脓毒症肺损伤时AEC外泌体的分泌情况；体内外抑制LPS诱导的AEC外泌体的分泌，观察其对减轻AM炎症反应的效果及对脓毒症肺损伤的保护作用；采用基因芯片结合前期miRNAs测序结果，筛选出LPS诱导的AEC外泌体中导致AM炎症反应的关键miRNAs及其靶基因，揭示外泌体介导AEC与AM间炎症信息的传递方式，探讨其促进脓毒症肺损伤的机制。

脓毒症肺损伤以上皮屏障遭到破坏，肺泡巨噬细胞（AM）聚集活化为主要特征，进而导致肺内失控性炎症反应，目前尚无有效的治疗策略。研究表明外泌体与AM炎症反应密切相关，但肺泡上皮细胞（AEC）来源的外泌体在脓毒症肺损伤中的作用尚不清楚。本项目采用盲肠结扎穿刺术（CLP）建立大鼠脓毒症肺损伤模型。通过气道滴入外泌体抑制剂GW4869来明确外泌体对脓毒症肺损伤的影响。利用超速离心法提取LPS刺激肺泡上皮细胞系RLE-6TN培养上清液中的外泌体（LPS-Exo）并建立LPS-Exo内miRNA表达谱，通过体内外实验阐明AEC来源的外泌体对AM活化及脓毒症肺损伤的作用机制。结果发现：（1）GW4869的干预显著减轻CLP术引起的脓毒症肺损伤；（2）体外提取的LPS-Exo可被AM摄取并活化AM，引起炎症反应；（3）经气道滴入LPS-Exo引起大鼠肺内炎症反应和通透性改变；（4）miR-92a-3p在LPS-Exo中富集并传递至AM，抑制miR-92a-3p可显著减弱LPS-Exo的促炎效应。其机制可能是miR-92a-3p引起PTEN的下调和NF-κB信号通路的激活，从而激活AMs以及肺部炎症。基于以上结果，我们得出结论：ACE来源的外泌体通过miR-92a-3p活化AM，引起肺内炎症反应。本项目揭示了外泌体源性miR-92a-3p介导受损AEC与AM间的信息交流，这有望成为脓毒症肺损伤的早期诊断标记物，为临床治疗提供新靶点。