miR-155上调自噬对重症急性胰腺炎小鼠肠黏膜屏障保护作用的实验研究

Secondary bacterial infection was the most serious complication of severe acute pancreatitis. The intestine was the the main source of the bacterial infection.Our preliminary studies shows that the uncontrolled release of inflammatory factors was the main reason of intestinal mucosa barrier damage.New research finds that inflammatory and caspase-1 activation are inhibited by autophagy, leading to decrease IL-1β release. Moreover, autophagy is negatively regulated by TAB2; up-regulation of miR-155 can down-regulate TAB2 expression. 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 the epithelial cells of intestinal mucosa might provide new strategy to block release of inflammatory mediators during severe acute pancreatitis. In this project, we will explore whether upregulation of miR-155 in epithelial cells of intestinal mucosa by transfection effects TAB2 expression, autophagy, caspase-1 and inflammatory mediators in vitro. Furthermore, we will take advantage of mouse severe acute pancreatitis model to examine whether miR-155 intravenous injection by in vivo-jetPEI protect intestinal mucosa barrier against injury, to investigate potential mechanisms. The success of such strategy will provide a novelty approach to treat intestinal mucosa barrier damage induced by severe acute pancreatitis.

胰腺继发感染是重症急性胰腺炎（SAP）最严重的并发症,肠道是引发胰腺继发感染的主要来源。本课题组前期研究表明,体内炎症因子失控性释放是SAP肠粘膜屏障损伤的主要原因，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，miR-155能靶向沉默TAB2蛋白的表达。我们推测通过在肠上皮细胞内过表达miR-155来上调自噬，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入肠上皮细胞，分析它对TAB2/自噬/炎性体和炎症介质的影响，并将in vivo-jetPEI介导的miR-155静脉注入SAP小鼠模型中，探讨对SAP小鼠肠粘膜屏障损伤保护作用及其机制，为临床上基因干预SAP肠粘膜屏障损伤的可行性提供依据。

在本次研究中，我们用雨蛙肽联合脂毒素（LPS）以及精氨酸成功构建重症急性胰腺炎小鼠模型，通过经典的自噬抑制剂3-MA以及自噬诱导剂调节自噬水平观察自噬对SAP模型的影响，结果发现抑制自噬受损的形成可使胰腺以及肺部病理损伤显著缓解，胰腺中NF-κB 以及caspase-1-IL-1β信号通路的激活也明显下调，这表明自噬药物抑制自噬功能受损通过NF-κB 以及caspase-1-IL-1β信号通路从而影响AP的预后。.在明确了自噬与胰腺炎的关系之后，我们进一步探讨了miR-155、自噬与胰腺炎之间的关系；首先我们通过腺病毒影响小鼠中miR-155的表达，发现沉默miR-155可改善AP小鼠中胰腺和肺损伤，过表达miR-155可以加重小鼠胰腺损伤，而予以自噬抑制剂能够缓解由miR-155介导的胰腺进一步损伤，其可能机制为调节自噬靶向蛋白TAB2，从而减少自噬受损和降低胰腺炎症反应。.此外，我们还发现在胰腺炎小鼠肠道中miR-155明显增加，因此我们推断miR-155可能在介导胰腺炎所致肠屏障破坏中起作用，利用特异性腺病毒影响肠道中miR-155的表达，发现miR-155过表达时胰腺炎小鼠肠屏障破坏更明显，炎症水平明显增加，利用16S高通量测序检测小鼠结肠组织肠道菌群的变化时，同样发现过表达miR-155时肠道菌群多样性明显下降，而干扰miR-155的表达时，胰腺炎肠屏障破坏、肠道炎症水平明显缓解，肠道菌群多样性明显上升。因此，我们可以发现miR-155不仅可以影响胰腺本身，而且对胰腺炎所致肠道炎症水平以及肠道屏障的破坏有重要影响，而肠道微生物可能在其中发挥重要作用。