miR-155上调自噬对脓毒症小鼠肺保护作用的实验研究

Treatment of lung injury induced by sepsis is limited by excessive activation of macrophage, resulting in uncontrolled release of inflammatory mediators. Our recent studies and those from others have shown: (1) miR-146a efficiently inhibits TLRs/NF-κB pathway and attenuates lung injury; (2) Inflammasome and caspase-1 activation are inhibited by autophagy, leading to decrease IL-1β release. Moreover, autophagy is negatively regulated by TAB2; (3) More importantly, up-regulation of miR-155 can down-regulate TAB2 expression. (4) Finally, we have strong evidence that activation of macrophages by LPS induced miR-155 overexpression and enhanced autophagy. Based on these findings, we hypothesize that overexpression of miR-155 in alveolar macrophages might provide new strategy to block release of inflammatory mediators during sepsis in lung. To this end, we will explore whether upregulation of miR-155 in alveolar macrophages by transfection effects TAB2 expression, autophagy, caspase-1 and inflammatory mediators in vitro. Furthermore, we will take advantage of mouse lung injury model induced by sepsis to examine whether miR-155 tracheal instillation by in vivo-jetPEI protect lung against sepsis induced injury, to investigate potential mechanisms. The success of such strategy will provide a novelty approach to treat sepsis induced lung injury.

肺内巨噬细胞过度活化，失控性释放炎症介质，是脓毒症肺损伤的根本原因。本课题组前一研究表明，miR-146a可以抑制TLRs/NF-κB炎症通路，减轻肺损伤。最新研究发现自噬能抑制炎性体及其关键效应蛋白caspase-l的活化，减少IL-1β等炎症因子的释放，而自噬受TAB2的负性调控，值得关注的是，miR-155能靶向沉默TAB2蛋白的表达。我们推测在肺泡巨噬细胞内过表达miR-155，有可能成为抑制炎症介质释放的新途径。我们的前期工作已证实LPS刺激肺泡巨噬细胞后miR-155表达上调、细胞自噬增加。本项目拟体外转染miR-155入肺泡巨噬细胞，分析它对TAB2/自噬/炎性体及caspase-l和炎症介质的影响，并将专门用于肺内转染的in vivo-jetPEI介导的miR-155气管滴入脓毒症小鼠肺损伤模型中，探讨其对小鼠脓毒症肺损伤保护作用及其机制，为治疗脓毒症肺损伤提供新途径。

肺内巨噬细胞过度活化，失控性释放炎症介质，是脓毒症肺损伤的根本原因。最新研究发现自噬能抑制炎性体及其关键效应蛋白Caspase-1的活化，减少IL-1β等炎症因子的释放，而自噬受TAB2的负性调控，值得关注的是，miR-155能靶向沉默TAB2蛋白的表达。本项目通过体内外转染miR-155 mimic或inhibitor，探讨miR-155对脓毒症肺损伤的保护作用及机制，结果发现：（1）miR-155的表达在脓毒症肺损伤患者的肺泡灌洗液中、盲肠结扎穿孔术诱导的脓毒症肺损伤小鼠模型肺组织中、内毒素刺激的NR8383肺泡巨噬细胞中的表达均增加，自噬水平在小鼠和细胞模型中也增加。（2）miR-155 mimic转染的小鼠模型中观察到肺组织炎症表现减轻，肺组织W/D减小，BALF中TNF-α、IL-1β含量减少；miR-155 mimic转染的细胞模型中也观察到了相似的结果；而细胞转染miR-155 inhibitor观察到Caspase-1表达上调、炎症因子水平增加。（3）在肺泡巨噬细胞中转染miR-155 mimic后，TAB2蛋白表达降低，自噬水平增加；在细胞中转染miR-155 inhibitor后TAB2蛋白表达升高，自噬水平下降。综上，miR-155可减轻脓毒症肺损伤炎症反应，其机制可能为miR-155通过靶向抑制TAB2，上调自噬。