[Role and mechanism of lipopolysaccharide induced exosome in the pathogenesis of acute lung injury].

Objective: To explore the effect and mechanism of exosome derived from lipopolysaccharide (LPS)-induced mouse macrophage (RAW264.7) on acute lung injury. Methods: RAW264.7 were cultured in vitro and divided into 2 groups: control group and LPS-induced group. The exosomes were extracted from the two groups of cell supernatant by ultracentrifugation and classified into 2 groups: C-EXO group and LPS-EXO group. In vivo, random allocation was used to averagely divide the eighteen male C57BL/6 mice into 3 groups: control group, EXO-control group and EXO-LPS group. All mice were sacrificed after 12 h. The lung tissue was used for HE staining to assess the degree of acute lung injury as well as immunohistochemical staining for interleukin (IL) -1β and tumor necrosis factor (TNF)-α. The tissue protein expression levels of IL-1β, TNF-α, β-catenin, E-cadhein, ZO-1 and Occludin were measured by Western blot. In vitro, alveolar type Ⅱ epithelial cells (MLE-12) were cultured and divided into 3 groups: C-control group, EXO-control-induced group and EXO-LPS-induced group. The tissue protein expression levels of IL-1β, TNF-α, and Occludin were measured by Western blot after 12 h. Results: The two samples of C-EXO group and LPS-EXO group was proved to be exosomes. Under a light microscope, the lung tissue of EXO-LPS group showed inflammatory cell infiltration, hemorrhage, interstitial and alveolar edema, and the thickness of alveolar septum. The tissue protein levels of IL-1β and TNF-α in EXO-LPS group were obviously higher than the control group, EXO-control group (1.331±0.203 and 0.274±0.018, 0.892±0.074; 0.800±0.096 and 0.596±0.025, 0.441±0.061; all P<0.05). While the tissue protein levels of Occludin showed the opposite phenomenon (0.251±0.021 and 0.862±0.029, 0.453±0.013; all P<0.05). In vitro, Compared with the C-control group and the EXO-control-induced group, the expression levels of IL-1β and TNF-α increased significantly in the EXO-LPS-induced group (0.900±0.033 and 0.320±0.030, 0.661±0.028; 0.739±0.045 and 0.151±0.024, 0.360±0.037; all P<0.05). whereas the protein levels of Occludin expression were reversed in MLE-12 (0.585±0.082 and 0.941±0.090, 0.732±0.083; all P<0.05). Conclusion: Exosomes derived from LPS-induced RAW264.7 can induced the acute lung injury by affecting barrier function, which probably is related to the low degree of Occludin in alveolar type Ⅱ epithelial cells.目的： 探讨脂多糖诱导巨噬细胞(RAW264.7细胞)释放的外泌体在小鼠急性肺损伤中的作用及其机制。 方法： 体外培养RAW264.7细胞，设置对照组和脂多糖刺激组，超速离心法提取两组细胞的外泌体，分别命名为对照EXO组和脂多糖EXO组。(1)体内实验：将18只C57BL/6雄性小鼠按随机数字表法随机均分成正常对照组、正常外泌体组、脂多糖刺激外泌体组各6只，造模12 h后放血处死，取肺组织行HE染色；行白细胞介素(IL)－1β和肿瘤坏死因子(TNF)－α的免疫组化评价；Western印迹检测各组IL－1β、TNF－α、β－链蛋白、E－钙黏蛋白、紧密连接蛋白1(ZO－1)和封闭蛋白的相对表达量；行组织切片免疫荧光对比各组间封闭蛋白的表达差异。(2)体外实验：将磷酸缓冲盐溶液(PBS)、对照EXO组和脂多糖EXO组外泌体与小鼠Ⅱ型肺泡上皮细胞(MLE－12细胞)共培养，设置空白对照组、对照EXO刺激组、脂多糖EXO刺激组。12 h后检测胞中IL－1β、TNF－α和封闭蛋白的相对表达量。 结果： 对照EXO组和脂多糖EXO组鉴定为外泌体。(1)体内实验：脂多糖刺激外泌体组肺组织出现肺泡腔及间质内广泛炎症细胞浸润，肺泡腔内有渗出液，肺泡隔增厚，肺泡及间质充血、出血，部分肺泡塌陷、不张等肺损伤表现；脂多糖刺激外泌体组肺组织中IL－1β和TNF－α蛋白相对表达量均显著高于正常对照组、正常外泌体组(1.331±0.203比0.274±0.018、0.892±0.074；0.800±0.096比0.596±0.025、0.441±0.061；均P<0.05)，脂多糖刺激外泌体组肺组织中封闭蛋白相对表达量均显著低于正常对照组、正常外泌体组(0.251±0.021比0.862±0.029、0.453±0.013；均P<0.05)。(2)体外实验：脂多糖EXO刺激组中IL－1β和TNF－α蛋白相对表达量均显著高于空白对照组、对照EXO刺激组(0.900±0.033比0.320±0.030、0.661±0.028；0.739±0.045比0.151±0.024、0.360±0.037；均P<0.05)，脂多糖EXO刺激组封闭蛋白相对表达量均显著低于空白对照组、对照EXO刺激组(0.585±0.082比0.941±0.090、0.732±0.083；均P<0.05)。 结论： 脂多糖诱导的RAW264.7细胞释放的外泌体导致小鼠发生急性肺损伤，且与肺泡Ⅱ型上皮细胞封闭蛋白的下调密切相关。.