PRMT6介导CIRP释放调控程序性坏死在肠缺血再灌注损伤中的作用及机制

Intestinal ischemia reperfusion (I/R) injury is a common perioperative complication in surgical patients. Our previous studies have confirmed that necroptosis is one of the mechanisms of intestinal I/R injury. Preliminary experiments showed that compared with wild type mice, the intestinal mucosa of PRMT6-/- mice showed slighter injury and decreased proportion of TUNEL positive cells after intestinal I/R, which suggests that necroptosis of intestinal epithelial cells might be inhibited. Bioinformatics analysis suggests that RPMT6 may catalyze arginine methylation of cold-induced RNA binding protein (CIRP). The translocation from nucleus to cytoplasm and the release into blood of CIRP are regulated by arginine methylation modification. The binding of extracellular CIRP and TLR4 induced necroptosis. We speculate that PRMT6 regulates necroptosis of intestinal epithelial cells by mediating the release of CIRP into blood during intestinal I/R. We will set up intestinal I/R model of gene knockout mice and oxygen-glucose deprivation/reoxygenation model of cell lines to explore the regulatory effect of PRMT6 on necroptosis of intestinal mucosal epithelial cells and the relationship between the release of CIPR into blood mediated by PRMT6 and necroptosis, and further explore the mechanism of PRMT6-mediated translocation and release of CIPR. The results will help to elucidate the molecular mechanism of intestinal I/R injury and provide new ideas and targets for the prevention and treatment of intestinal I/R injury.

肠缺血再灌注（I/R）损伤是手术患者围术期常见并发症。我们既往研究证实程序性坏死是肠I/R损伤机制之一。预实验发现PRMT6-/-小鼠肠I/R损伤较野生型小鼠减轻，肠组织TUNEL阳性细胞比例下降，提示程序性坏死受到抑制。生物信息学分析提示RPMT6可能催化冷诱导RNA结合蛋白（CIRP）的精氨酸甲基化。文献报道CIRP从细胞核转位至细胞质并释放入血的过程受精氨酸甲基化调控，而胞外CIRP能结合TLR4，介导细胞程序性坏死。我们推测：PRMT6通过介导CIRP释放入血进而调控肠I/R所致肠上皮细胞程序性坏死。本项目将利用基因敲除小鼠肠I/R模型和细胞氧糖剥夺/复氧模型，探究PRMT6对肠上皮细胞程序性坏死的调控作用以及PRMT6介导CIPR释放与程序性坏死的关系，并探讨PRMT6调控CIPR转位释放的具体机制。研究结果有助于阐明肠I/R损伤的分子机制，为肠I/R损伤的防治提供新思路和靶点。

肠道缺血再灌注（肠I/R）损伤是围术期常见的病理生理过程，可见于肠系膜缺血性疾病、小肠移植、心血管深低温停循环手术、肝移植手术门静脉血流阻断及失血性休克等过程。肠I/R不仅导致肠本身的损伤，还可释放肠粘膜上皮细胞内的致炎物质入血，造成远程器官损伤。本项目从Bax/Bcl2介导的细胞凋亡调控入手探讨了肠缺血再灌注损伤的新机制。取得的研究结果包括：（1）研究证实了肠I/R后HMGB 1释放、肺中性粒细胞浸润和NETs产生是小鼠肠I/R诱导的急性肺损伤的重要机制。我们发现小鼠肠I/R后肺上皮细胞NETosis增加，通过DNase I降解NETs和PAD 4抑制剂抑制NETosis可以减轻肠I/R诱导的急性肺损伤。此外，通过使用Ly6G中和抗体预处理消耗中性粒细胞以及HMGB1中和抗体都可以减轻肠I/R后肺上皮细胞NETosis，MyD88敲除也可以减轻肠I/R后肺上皮细胞NETosis，减轻肺损伤。综上，肠I/R后坏死肠上皮细胞释放HMGB 1，通过激活TLR4-MyD 88信号通路激活肺中性粒细胞NETosis，是肠I/R诱导急性肺损伤的重要机制。（2）研究证实了Bax/Bcl-2介导的细胞凋亡参与了小鼠肠I/R损伤。在Caco-2细胞系离体OGD/R模型中，我们发现了Bax/Bcl-2介导的细胞凋亡发挥了重要作用，而Parkin则通过下调Bax蛋白水平同时上调Bcl-2水平，抑制Bax/Bcl-2介导的细胞凋亡途径发挥保护作用。FOXO3a作为转录因子，可以上调Parkin的转录水平，而PRMT6则可以通过与FOXO3a结合增加其活性。综上，PRMT6通过激活FOXO3a进而上调Parkin表达水平，抑制Bax蛋白水平的同时提高Bcl-2蛋白水平，减轻缺血再灌注导致的肠上皮细胞凋亡。本项目为肠I/R损伤的防治提供了新的切入点。同时，研究结果提示了PRMT6抑制使用的潜在风险，为PRMT6抑制剂的临床应用适应证及禁忌证提供了一定的理论参考。