HMGB1-LncRNA-EPS/NLRP3炎性调控网络介导肠缺血再灌注肠损伤后巨噬细胞M1型极化的机制研究

Macrophage is documented to be an important mediator involved in local immune regulation after intestinal ischemia/reperfusion (I/R) injury. Our previous investigation has demonstrated that intestinal I/R results in significant macrophages activation and a switch from M2 to M1 macrophages, while a reversing intestinal I/R-induced macrophage switch is effective in ameliorating intestinal injury and improving intestinal function. But the underlying mechanisms are still unclear. We also found that HMGB1 released after intestinal I/R led to the activation of HMGB1/TLR4 signaling. It is known that HMGB1 mediates NLRP3 activation, which is subsequently related to the regulation of macrophage polarization. Meanwhile, our preliminary study has found that the expression of LncRNA-EPS in macrophage is down-regulated following HMGB1 stimulus, and LncRNA-EPS interference leads to the change of NLRP3 expression. Therefore, we aimed to explore the underlying new mechanisms that HMGB1-LncRNA-EPS/NLRP3 inflammatory network mediated M1 polarization of macrophages after intestinal I/R injury by applying in vivo and in vitro models.

巨噬细胞是肠黏膜免疫屏障的组成部分，在肠缺血再灌注（I/R）损伤后的局部免疫功能调节中起重要作用。我们近期发现肠I/R后肠巨噬细胞激活并向M1型转变，而逆转这种功能极化能改善黏膜屏障功能障碍，具体调控机制尚不清楚。我们还发现肠I/R后释放的HMGB1促使胞内HMGB1/TLR4信号通路活化。已知HMGB1能介导NLRP3炎性小体激活并参与巨噬细胞极化调控。预实验发现HMGB1能下调巨噬细胞LncRNA-EPS表达，同时干扰EPS表达可引起NLRP3蛋白表达变化。据此，我们拟在离体、在体研究中使用一系列分子生物学手段证实肠I/R损伤后HMGB1与巨噬细胞表面TLR4受体结合后传导胞内信号下调LncRNA-EPS表达，通过转录调控致NLRP3炎性小体活化后促进巨噬细胞向M1型极化导致肠道免疫失控，同时释放炎症产物介导黏膜屏障功能障碍。本项目期望揭示肠I/R后肠巨噬细胞功能极化调控的新机制。

肠缺血再灌注（I/R）常出现在创伤、休克、重症感染及腹主动脉瘤手术等临床现象中。肠I/R后肠黏膜屏障及肠外多器官损伤的发生机制尚不明确。我们通过动物实验发现肠道缺血损伤后肠黏膜中miRNA-387的表达下调失去对靶基因Caspase-3的抑制，促使黏膜上皮细胞凋亡Apoptosis增加，进而介导肠组织损伤；而联合抑制RIP1/RIP3介导的程序性坏死Necroptosis及PARP-1介导的Parthanatos可显著改善肠I/R后的肠损伤。上述研究成果从不同的肠黏膜上皮细胞死亡方式调控揭示了肠道机械屏障结构和功能损伤的新机制，为临床寻找防治肠I/R损伤可供干预的靶点提供了扎实的理论基础。.肠道作为多器官功能不全综合症的“启动”和“枢纽”器官，肠I/R损伤后导致肠外器官损伤的机制尚不清楚。我们通过动物实验发现：①肠道I/R损伤可以导致急性肝脏损伤，以再灌注后6小时最为显著。肠道大肠杆菌Escherichia coli肝脏内异位、肝细胞中HMGB1细胞质内移位、肝细胞发生Necroptosis、肝库普弗细胞和循环巨噬细胞向M1型巨噬细胞极化等机制介导了上述的病理生理过程。②肠I/R后可造成远程胰腺组织的损伤，该过程与肠黏膜上皮细胞和胰腺腺泡细胞均发生Necroptosis相关。③肠I/R后释放的HMGB1通过PERK-ATF4-CHOP通路激活肾脏细胞内质网应激介导了肾功能损伤。④肠I/R后肺组织丰富的毛细血管网聚集了大量中性粒细胞，并形成中性粒细胞胞外诱捕网（NETs）结构，在发挥抗感染免疫功能的同时介导了肠I/R后的肺组织损伤。HMGB1-Myd88信号通路在趋化和激活中性粒细胞形成NETs中发挥了重要的作用，而重组HMGB1蛋白可以加重肠I/R后的肺组织损伤。上述研究结果系统性探索了肠I/R损伤后导致肠外多器官损伤的相关机制并提出了可能的干预措施。