MiR-381-3p Knockdown Improves Intestinal Ischemia/Reperfusion Injury By Upregulating Nurr1

Background: Intestinal ischemia/reperfusion(I/R)injury is a common clinical pathophysiological process.Intestinal I/R injury occurs not only in the intestinal disease and the mesenteric vascular disease,but also secondary to trauma,shock,organ transplantation and other clinical pathological processes.Intestinal I/R leads to the damage of intestinal mucosal barrier,migration of bacteria and endotoxin,which can trigger systemic inflammatory response syndrome(SIRS).Severe cases suffer from multiple organ dysfunction syndrome(MODS).Therefore,it has important clinical significance to improve the intestinal barrier function after intestinal I/R injury.Nuclear receptor-related factor 1(Nurr1)is a member of the nuclear receptor superfamily.Our previous results showed that Nurr1 expression was reduced after intestinal I/R injury and was associated with proliferation of intestinal epithelial cells.Several stages of proliferation,migration and differentiation of intestinal epithelial cells control the intestinal barrier function.Here,our research shows that microRNAs(miRNAs)regulate the differentiation of dopaminergic neurons by binding to the 3'UTR region of Nurr1.Recent studies revealed that miRNAs regulate the intestinal I/R injury by binding to the 3'UTR region of the target gene.Therefore,we hypothesized that Nurr1 is a key molecule for intestinal barrier repair after intestinal I/R injury.Nurr1 expression during reperfusion may be regulated by miRNA.This miRNA regulates the expression of Nurr1 and regulates restoration of intestinal barrier function after injury,which in turn affects small intestinal I/R injury.In this study,we used a microarray chip and several bioinformatics databases to predict the targeted regulatory relationship between miR-381-3p and Nurr1.We intervene with miR-381-3p and Nurr1 to elucidate that miR-381-3p regulates the Nurr1 pathway.The role of intestinal mucosal barrier repair following small intestine I/R injury provides a new therapeutic target for intestinal mucosal barrier repair after I/R injury.Objective: To investigate the effect of Nurr1 on the improvement of intestinal barrier function after intestinal I/R injury.To explore the protect effects and mechanisms of miR-381-3p on intestinal I/R injury by targeting Nurr1.Methods: 1.These healthy male C57 mice were randomly divided into four following groups(n=8 per group):(1)sham group;(2)sham+C-DIM12 group;(3)I/R group;(4)I/R+C-DIM12.C-DIM12(50 mg/kg body weight)was administered by gavage at four hours prior to surgery.Midline laparotomy was performed in the sham-operated mice.All of the mouse superior mesenteric arteries in the sham-operated group were treated with isolated but not clamped.Midline laparotomy was performed in the I/R group mice.All of the mouse superior mesenteric arteries was identified,isolated and clamped.After 45 min of ischemia,the vascular clamp was removed from the artery to allow reperfusion 4h.The small intestine tissue was collected and stored in appropriate condition(-80?)for experiment.The pathological changes of small intestine were treated with H&E staining.Western-blot was used to detect the intestinal proteins expression,such as occludin,ZO-1.2.Adult male C57 mices were randomly divided into four following groups(n=8,per group):(1)sham+LNA-NC group;(2)sham+LNA-381 group;(3)I/R+LNA-NC group;(4)I/R+LNA-381 group.we injected mice with 2 mg/kg locked nucleic acid-modified antisense oligonucleotides targeting miR-381-3p(LNA-381)or LNA-NC oligonucleotides via the tail vein at 12 h before ischemia.The model of intestinal I/R was performed with above protocol.Small intestine tissues were collected and stored in suitable condition(-80?)for experiments.Histopathological changes of small intestine was detected by H&E staining.Western-blot was used to detect the intestinal proteins expression,such as Nurr1,p21,occludin and ZO-1.3.(1)Caco-2 or IEC-6 cells were used to construct hypoxia/reoxygenation(H/R)model to stimulate intestinal I/R injury.Those cells were divided into 4 following groups,including Control+ant-NC;Control+ant-381;I/R+ant-NC;I/R+ant-381.Fluorescence intensity of FD4 was used to detect the permeability of intestinal epithelial cell.Western-blot was carried out to explore the expression levels of Nurr1,p21,occludin and ZO-1.(2).Caco-2 or IEC-6 cells were used to construct hypoxia/reoxygenation(H/R)model to stimulate intestinal I/R injury.Those cells were divided into 5 following groups,Control+ant-NC+si-control group;H/R+ant-NC+si-control group;H/R+ant-381+si-control group;H/R+ant-NC+si-Nurr1 group;H/R+ant-381+si-Nurr1 group.The H/R groups were subjected to hypoxia for 12 hours and reoxygenation for 6 hours to establish H/R model.In addtion,Fluorescence intensity of FD4 was used to detect the permeability of intestinal epithelial cell.Western-blot was carried out to explore the expression levels of Nurr1,p21,occludin and ZO-1.Results: 1.Compared to Sham group,mice in I/R group had obvious intestinal pathological damage and the down-regulation of occludin,ZO-1 expression.The intestinal pathological damage of I/R+C-DIM12 group was significantly alleviated and the expression of occludin and ZO-1 was increased when compared to I/R group.2.According to bioinformatics databases and microarray analysis,miR-381-3p can target 3'UTR region of Nurr1.miR-381-3p decreased Nurr1 expression and wild-type Nurr1 3'UTR activity;miR-381-3p knockdown upregulated Nurr1 expression;miR-381-3p had not effect on Nurr1 mRNA expression and mutant SIRT1 3'UTR activity.3.Compared with Control+ant-NC group,the intestinal epithelial permeability increased and the expression of Nurr1,occludin and ZO-1 downregulated and the expression of p21 increased in H/R+ant-NC group;ant-381 alleviated intestinal epithelial permeability,the upregulation of Nurr1,occludin and ZO-1 expression and the downregulation of p21 expression after H/R injury.4.Compared with Sham+NC group,I/R+NC group showed significant pathological damage of small intestine and the downregulation of Nurr1,occludin and ZO-1 expression.Compared with I/R+NC group,the intestinal pathological damage was significantly alleviated,the expression of Nurr1,occludin and ZO-1 was increased and the expression of p21 was decreased in I/R+LNA-381 group.5.The permeability of H/R+ant-381+si-control intestinal epithelial cells was significantly lower than that of H/R+ant-NC+si-control group.There was no significant change in the intestinal epithelial permeability of H/R+ant-381+si-Nurr1 when compared to the H/R+ant-NC+si-control group.Compared with H/R+ant-NC+si-control group,the expression of Nurr1,occludin,ZO-1 increased and the expression of p21 decreased in H/R+ant-381+si-control group.There was no significant change in the expression of Nurr1,occludin,ZO-1 and p21 in H/R+ant-381+si-Nurr1 group when compared to H/R+ant-NC+si-Nurr1 group.Conclusion: 1.Nurr1 plays an important role in the epithelial repair after intestinal I/R injury.2.miR-381-3p knockdown improves intestinal I/R injury,which showed by decreasing the permeability of intestinal epithelium,decreasing the intestinal histopathologic injury,improving the expression of intestinal barrier protein.