Nurr1/p21通路在肠缺血再灌注损伤修复中的作用机制及miR-381-3p的调控作用

Intestinal injury and repair are two co-existing pathophysiological processes during the intestinal ischemia/reperfusion (II/R), which has the complex mechanism. If the mucosa regeneration cannot repair the damage, it will lead to intestinal barrier dysfunction, and even induce SIRS and MODS. Nuclear receptor related factor 1 (Nurr1) is an important transcription factor that regulates tissue regeneration after injury. For the first time, we found that Nurr1 was differentially expressed in II/R intestinal tissue in mice and positively correlated with the mucosal regeneration; Nurr1 knockout could up-regulate expression of p21 and inhibit intestinal epithelial cell proliferation; Expression of Nurr1 was negatively correlated with miR-381-3p, and bioinformatics predicted that Nurr1 is a target of miR-381-3p. Accordingly, we proposed that regulating Nurr1/p21 by miR-381-3p plays an important role in intestinal mucosal repair and regeneration after II/R. The aim of this study is to explore the effect of Nurr1/p21 pathway in the intestinal regeneration after II/R both in vivo and in vitro, and to investigate the regulating effect of miR-381-3p on Nurr1/p21 signal pathway in the regeneration process of II/R.

肠缺血再灌注 (II/R) 后肠道的损伤和修复是两个并存的病理生理过程。当肠道黏膜的再生不足以修复损伤会引发肠道屏障功能障碍，进而产生SIRS和MODS。核受体相关因子1（Nurr1）是调控组织损伤后再生的重要转录因子。我们首次发现，Nurr1在II/R后肠组织中呈差异性表达，与损伤后肠黏膜再生呈正相关；敲除Nurr1可上调p21的表达并抑制肠上皮细胞增殖；Nurr1的表达与miR-381-3p负相关；生物信息学预测miR-381-3p靶向Nurr1。据此提出，在II/R后再生修复过程中，可能存在Nurr1-p21信号通路，miR-381可调控该通路对小肠黏膜上皮的再生修复产生影响。本项目拟结合动物、离体细胞模型，运用分子生物学技术，探讨Nurr1/p21通路在II/R后再生中的作用机制以及miR-381-3p靶向该通路对肠黏膜再生修复的作用。

肠缺血再灌注（II/R）是引发全身炎症反应综合征（SIRS）或多器官功能障碍综合征（MODS）的重要始动因素。其发病机制复杂，肠上皮细胞黏膜屏障功能损伤是II/R损伤过程中重要的发病机制。我们前期研究发现，受体相关因子1（Nurr1）是调控组织损伤后再生的重要转录因子，其在II/R后肠组织中呈差异性表达，与损伤后肠黏膜增殖水平呈正相关；敲除Nurr1可上调p21的表达并抑制肠上皮细胞增殖。我们提出假说：在II/R后再生修复过程中，可能存在Nurr1-p21信号通路，miR-381可调控该通路对小肠黏膜上皮的再生修复产生影响。本研究采用野生型（Nurr1-WT）及条件性 Nurr1 基因敲除（Nurr1-KO）小鼠建立肠缺血再灌注（II/R）及IEC-6细胞缺氧/复氧（H/R）实验模型，阐明Nurr1调控p21表达在II/R损伤后再生修复的作用机制；采用 miR-381 的激动剂和抑制剂对 Nurr1 进行干预，探讨 miR-381 对 Nurr1-p21通路的调控作用及机制。结果发现：（1）Nurr1在缺血小肠的再灌注早期表达逐渐降低，而后表达逐渐恢复，Nurr1的表达抑制可引起I/R后小肠粘膜细胞增殖受阻。（2）通过生物信息学预测结合实时定量PCR，证实了miR-381-3p可直接靶向结合Nurr1并在肠上皮细胞中负向调控Nurr1表达。（3）抑制miR-381-3p可在体内外减少肠上皮细胞通透性、增加跨膜电阻，其保护作用可能与下调p21的表达、减轻细胞周期阻滞、促进细胞增殖有关。此外，下调miR-381-3p还可减轻小肠组织病理学损伤，促进肠上皮细胞增殖，减轻远隔肺肝损伤，减轻全身炎症反应，改善动物生存率。（4）抑制miR-381-3p对肠I/R的保护作用与其对Nurr1的调控紧密相关。该研究首次证实miRNA在肠I/R损伤过程中对肠上皮细胞增殖与再生的调控作用，抑制miR-381-3p通过上调Nurr1表达促进肠上皮细胞增殖，进而缓解肠I/R多器官损伤。