| Objective:Irritable Bowel Syndrome with Diarrhea(IBS-D)is the most common functional bowel disease.Acupuncture treatment of IBS-D has definite curative effect and outstanding advantages,but the mechanism of acupuncture effect needs to be deepened.Previous studies have found that electroacupuncture(EA)can alleviate the intestinal symptoms and psychological abnormalities of IBS-D.The expression of corticotropin releasing factor receptor 1(CRF-R1)and the activity of mast cell(MC)in the colon tissue of IBS-D are decreased.On the basis of previous studies,this study was to observe whether EA regulates the expression of MC-derived exosome(MC-EXO)microRNA(miRNA)through CRF-R1 and MC pathways,which can repair intestinal epithelial barrier function and improve IBS-D symptoms.To explore the effect mechanism of EA on IBS-D.Methods:1.Rats were randomly divided into blank group,model group,EA group,EA+Ucn1(CRF-R1 agonist)group and EA+C48/80(mast cell agonist)group.Except for the blank group,IBS-D model was established by gavage with senna leaf extract and chronic unpredictable mild stress(CUMS),and the model was established for 14 days.In EA group,"Tian Shu"(ST25),"Zusanli"(ST36)and "Taichong"(LR3)points were selected,and the frequency of EA was 2 Hz/15Hz,the intensity was 0.5mA,once a day,20 min each time,for 14 consecutive days.EA+Ucn1 group and EA+C48/80 group were injected with agonist for 30 min before EA intervention.The changes of symptoms in rats were evaluated by fecal score,visceral pain threshold and behavioral indexes,pathological changes were observed by HE staining in colon,and the expressions of corticotropin releasing factor(CRF)and CRF-R1 mRNA were detected by RT-PCR.The status of mast cells was observed by transmission electron microscope and immunohistochemistry,and the intestinal epithelial barrier function was detected by ELISA and Western Blot.The effects of EA on IBS-symptoms,CRF-R1,mast cell pathway and intestinal epithelial barrier function were analyzed.2.Rats were randomly divided into blank group,model group,EA group and EA+GW4869(exocrine inhibitor)group.Model preparation and EA intervention are the same as experiment.During the intervention period,EA+GW4869 group was injected intraperitoneally with GW4869 every day for 30 min,and then EA was performed.The content of serum DAO was detected by ELISA,and the expression of colon tight junction protein was observed by immunohistochemistry,and the effect of EA on improving intestinal epithelial barrier function after inhibiting exocrine secretion was observed.3.The groups were the same as the first and second parts.Percoll density gradient separation method was used to extract MCs from peritoneal lavage fluid,and ultra-high speed differential centrifugation method was used to separate MC-EXO.The MC-EXO miRNAs in blank group,model group and EA group were sequenced by Illumina Xten to screen the key MC-EXO miRNAs for EA regulation.GO and KEGG function enrichment analysis of key miRNAs was carried out through DAVID website.RT-PCR was used to verify the expression of key miRNAs in blank group,model group,EA group,EA+Ucn1 group,EA+C48/80 group and EA+GW4869 group.Finally,Spearman correlation analysis was made between the verified MC-EXO miRNA and the permeability of intestinal epithelial barrier,and the key MC-EXO miRNAs for EA to repair the intestinal epithelial barrier function of IBS-D was screened.4.Based on the results of the third part,Caco-2 cells were taken as the research object to construct the intestinal epithelial barrier cell model.miR-149-5p mimics and miR-22-5p mimics were used to transfect the intestinal epithelial cells,RT-PCR was used to detect whether the transfection was successful,and FD-40 permeability test and Western Blot were used to detect the intestinal epithelial barrier function.All the results were to verify the effect of key miRNAs on intestinal epithelial barrier.Results:1.Compared with the blank group,the Bristol score in the model group decreased(P<0.05),the visceral pain threshold score increased,and the OT%and the total distance of open field experiment decreased.A small number of inflammatory cells can be seen in the stroma of the lamina propria of colon in the model group.Compared with the model group,the Bristol score,visceral pain threshold,OT%and the total distance of open-field experiments in the EA group decreased significantly(P<0.05).CRF mRNA and CRF-R1 mRNA decreased significantly,and the expression of tryptase decreased(P<0.01).The content of serum DAO decreased,the expression of ZO-1,Claudin-1 and Occludin increased,and the MCs and tight junction structures were normal under transmission electron microscope.CRF-R1 agonist and mast cell agonist were used for pretreatment before acupuncture,which blocked the above acupuncture effect.2.Compared with the blank group,the serum DAO content,and the expressions of ZO-1,Claudin-1 and Occludin protein decreased significantly in the model group increased significantly(P<0.01).Compared with the model group,the content of DAO in EA group and EA+GW4869 group decreased,and the degree of decrease in EA+GW4869 group was more obvious than that in EA group(P<0.01).The expression of ZO-1,Claudin-1 and Occludin protein in EA group and EA+GW4869 group increased significantly,and the increase degree of EA+GW4869 was more obvious than that in EA group(P<0.01).3.Compared with the blank group,there are 9 differentially expressed miRNAs in the model group,of which 5 are up-regulated and 4 are down-regulated.Compared with the model group,there are 10 differentially expressed miRNAs in the EA group,all of which are down-regulated.After crossing Wayne diagram,three miRNAs were obtained:rno-miR-1-3p,rno-miR-22-5p and rno-miR-149-5p.RT-PCR results showed that the expression of rno-miR-22-5p and rnomiR-149-5p were consistent with the sequencing results,and were the same as those of intestine.4.After miR-149-5p mimics and miR-22-5p mimics were transfected into the intestinal epithelial cell model,the relative transmittance of FD-40 increased significantly,and the expressions of ZO-1,Claudin-1 and Occludin decreased significantly,with statistical significance.Conclusion:1.EA can reduce the expression of CRF and CRF-R1 and inhibit the activity of mast cells,thus improving the intestinal symptoms,anxiety and depression of IBS-D and repairing the intestinal epithelial barrier function.2.MiR-22-5p and miR-149-5p can decrease the expression of tight junction protein and increase the permeability of intestinal epithelial barrier.EA may decrease the expression of MC-EXO miR-22-5p and miR-149-5p and improve the intestinal epithelial barrier function of IBS-D. |
