| Inflammatory bowel disease(IBD)is a chronic nonspecific intestinal inflammatory disease,which mainly includes Crohn’s disease(CD)and ulcerative colitis(UC).The pathogenesis of IBD is related to genetic susceptibility,environmental factors and flora imbalance,which can lead to immune response dysfunction and local mucosal homeostasis imbalance,characterized by severe intestinal inflammation and tissue damage.Among them,the imbalance of microflora is considered to be an important trigger for the impairment of intestinal barrier function and the destruction of immune homeostasis,and it is also a decisive event leading to disease progression and chronicity.Therefore,restoring the balance of intestinal flora and reshaping intestinal microecology has important clinical significance for the remedial treatment of patients with IBD.At present,the effect of the treatment for correcting flora imbalance is uncertain,and its long-term tolerance and safety are not clear.Therefore,how to effectively reshape intestinal flora is still an important problem in the field of IBD therapy.Mesenchymal stem cells(MSCs)therapy is a promising new method for the treatment of IBD,mainly by inhibiting inflammatory response,regulating immune homeostasis and promoting the repair of intestinal mucosal barrier.At present,the studies of MSCs in the treatment of IBD mostly focused on promoting the proliferation and differentiation of regulatory T cells(Tregs)expressing transcription factor Foxp3 and inhibiting the activity of inflammatory T cells and the secretion of pro-inflammatory factors.Recent studies have found that MSCs not only improve intestinal inflammation,but also remodel the diversity and abundance of intestinal flora and adjust the proportion of bacteria in colitis mice.Although there is still insufficient evidence and the related mechanism has not been reported,it is speculated that remodeling intestinal flora may be another potential mechanism of MSCs in the treatment of IBD.Besides,compared with other tissue sources,human umbilical mesenchymal stem cells(HUMSCs)have many advantages,which can be isolated and cultured in vitro,easy to obtain,with stable biological performance,low immunogenicity,and without ethical controversy.Therefore,it is extremely suitable for clinical and basic research.The immune regulation of host intestinal mucosa plays an important role in maintaining the diversity of intestinal flora.It has been proved that the main role of immunoglobulin A(IgA)is to maintain the balance between host and flora,in which intestinal secretory IgA(s IgA)can specifically bind to intestinal bacteria,maintaining the structure and diversity of intestinal flora,preventing the adhesion of pathogenic bacteria to epithelial cells,neutralizing toxins and pathogens,limiting the growth and penetration of pathogenic bacteria,and maintaining intestinal homeostasis.It has been found that the presence of Foxp3~+Tregs in intestinal mucosa supports the transformation of IgA~+plasma cells,maintains the selective secretion of IgA in the intestine,and enhances the immune barrier,although the mechanism has not been fully elucidated.In the model of T cell metastatic colitis,Foxp3~+Tregs metastasis can inhibit colonic inflammation,induce the transformation of IgA~+plasma cells in the lamina propria of intestinal mucosa,promote the production of s IgA,and maintain the diversity of intestinal flora.The absence of Foxp3~+Tregs reduces the intestinal IgA response,reduces the number of beneficial bacteria and increases the number of harmful bacteria,thus aggravating the inflammatory response of the colon.It can be seen that Tregs-IgA response plays an important role in maintaining the balance of intestinal microecology.In summary,in view of the important role of Foxp3~+Tregs,whether the mechanism of MSCs remodeling intestinal flora is related to the regulation of intestinal Tregs-IgA response is worthy of further discussion.In this study,C57BL/6 mice were given dextran sulfate sodium(DSS)to establish IBD model.After intraperitoneal injection of HUMSCs,the mortality,body weight,colon length,disease activity index(DAI)and pathological score of colons were evaluated to explore the therapeutic effect of HUMSCs on colitis.The fecal microflora diversity of mice in each group was detected by16S r RNA high-throughput sequencing,and the microbial composition and abundance were determined at the phylum,genus and species level,and the differential core flora was analyzed to explore the effect of HUMSCs on intestinal flora of colitis mice.The mononuclear cells of lamina propria,mesenteric lymph nodes(MLN)and Peyer’s patches(PPs)of mice were further isolated.The ratio of Foxp3~+Tregs and IgA~+plasma cells in mononuclear cells of colitis mice was analyzed by flow cytometry to understand the effect of HUMSCs on the differentiation of Foxp3~+Tregs and IgA~+plasma cell.Immunohistochemical and ELISA methods were used to detect the secretion of IgA in intestinal tract and feces of mice.The proportion of IgA encapsulated bacteria was evaluated by flow cytometry to further clarify the effect of HUMSCs on Tregs-IgA response,in order to clarify the possible mechanism of HUMSCs remodeling intestinal flora.The experiment mainly includes the following three parts:Part One HUMSCs promote the repair of intestinal mucosal inflam-mation in mice with experimental colitis.Objective:To explore the protective effect of HUMSCs on intestinal mucosal inflammation in mice with experimental colitis induced by DSS.Methods:1)Cultured and passaged of HUMSCs.HUMSCs were isolated and purified by tissue separation and adhesion methods,the phenotype of HUMSCs was identified by flow cytometry,and the expression of positive markers of CD90,CD105,CD73,CD44,CD29 and negative markers of CD45and HLA-DR in HUMSCs were detected.2)The mouse model of experimental colitis induced by DSS was established and randomly divided into Control group,DSS+PBS group and DSS+MSCs group.3)The body weight,mental state and stool condition of mice were recorded every day,and the DAI score was performed.At the same time,the survival of mice was recorded every day.4)The changes of colonic length among different groups of mice were compared,and the repair of intestinal mucosal inflammation was evaluated by H&E staining.Results:1)The logarithmic phase P3 HUMSCs cultured in vitro were selected.Flow cytometry showed that the expression of CD90,CD105,CD29,CD44and CD73 was positive,while the expression of CD45 and HLA-DR was negative,suggesting that the cells were human umbilical cord mesenchymal stem cells,which met the identification criteria and could be used in follow-up experiments.2)The mouse model of experimental colitis induced by DSS was successfully constructed.3)After HUMSCs treatment,the weight loss of mice and the degree of hematochezia gradually reduced.Compared with DSS+PBS group,the body weight,DAI score and mortality of mise in DSS+MSCs group were significantly lower(all P<0.05);4)Compared with DSS+PBS group,the colon length of DSS+MSCs group was longer(P<0.001);5)Pathological score:Compared with DSS+PBS group,the colon structure of DSS+MSCs group was relatively intact,mononuclear cell infiltration was less,mucosal injury was milder,and histopathological score was significantly lower than that of DSS+MSCs group(P<0.001).Conclusions:HUMSCs have therapeutic effect on experimental colitis and can promote the repair of intestinal mucosal inflammation.Part Two HUMSCs remodel the structure and diversity of intestinal flora in mice with experimental colitisObjective:To observe the effect of HUMSCs on the structure and diversity of intestinal flora in mice with experimental colitis induced by DSS.Methods:1)Downloaded from the NCBI public database(https://www.ncbi.nlm.nih.gov/sra)on UC patients and healthy people feces 16 s r RNA sequencing data(SRP071201),further integration analysis group differences between flora and understand the changes of intestinal flora in patients with UC.2)The animal experiment group was the same as the first part.3)The feces of mice in each group were collected,and the genomic DNA were extracted and purified.4)High-throughput sequencing of DNA library was carried out with the help of Illumina Mi Seq second-generation sequencing technology platform,species annotation and abundance analysis were carried out based on QIIME2software,intestinal microfloraα-diversity andβ-diversity were analyzed,relative species abundance column chart was drawn,and LEf Se differential microflora analysis was performed to evaluate the changes of intestinal microflora abundance,composition and core microflora differences in mice with experimental colitis.Results:1)Through the integrated analysis of 16S r RNA sequencing database of clinical samples of UC patients,it was found that compared with healthy controls,UC patients had intestinal flora dysregulation,Firmicutes(39.44±2.88 vs 20.59±5.60,P<0.001),Bacteroidota(4.65±2.90 vs 28.72±10.82,P<0.001)were significantly reduced.2)βdiversity:Based on PCo A analysis and non-metric multidimensional NMDS scale analysis,the composition of intestinal microflora in DSS+PBS group changed significantly compared with Control group.HUMSCs intervention could reshape the intestinal microflora distribution in mice with colitis and was more similar to healthy control group.3)αdiversity:Compared with Control group,Chao1 index of species richness increased after DSS intervention,while Chao1 index decreased after HUMSCs treatment,which was closer to that of healthy control group,although the difference was not statistically significant(P>0.05);Compared with Control group,Simpson index(P<0.01)and Pielou＿e index(P<0.05)of DSS+PBS group were significantly increased,suggesting that DSS changed the diversity and evenness of intestinal flora in colonic contents of mice,while the treatment of HUMSCs reversed this change(P<0.05),and there was no significant difference between DSS+MSCs group and Control group(P>0.05).4)The analysis of species abundance showed that compared with Control group,the Firmicutes of DSS+PBS group significantly increased(P<0.001)and Bacteroidota significantly decreased(P<0.01).However,DSS+MSCs group reversed this change,decreased Firmicutes(P<0.01)and increased Bacteroidota(P<0.05).At genus level,Muribaculaceae in DSS+PBS group was significantly lower than that in Control group(P<0.01),while Lachnospiraceae＿NK4A136＿group and Clostridia＿UCG-014 were signifi-cantly higher than those in Control group(P<0.01).The use of HUMSCs restored the changes of Muribaculaceae(P<0.01)and Lachnospiraceae＿NK4A136＿group(P<0.05)induced by DSS.5)Through the LEf Se analysis of the core flora of each group,it was found that Clostridia class,Lachnospirales and Oscillospirales order,Lachnospiraceae and Oscillospiraceae family in DSS+PBS group,as well as Lachnospiraceae＿NK4A136＿group and Eubia＿rumantium＿group were the main differential bacteria leading to intestinal flora imbalance,while the treatment of HUMSCs down-regulated the level of these bacteria.Conclusions:1)HUMSCs remodeled the diversity and abundance of intestinal flora in mice with experimental colitis induced by DSS,and regulated the composition of intestinal flora.2)HUMSCs down-regulated the main differences in intestinal flora caused by DSS,and promoted the recovery of microecological balance in mice with experimental colitis.Part Three Regulatory effect of HUMSCs on intestinal Tregs-IgA res-ponseObjective:To observe the effect of HUMSCs on Tregs-IgA response in mice with experimental colitis induced by DSS,and to explore the possible mechanism of HUMSCs regulating intestinal flora.Methods:1)The animal experiment group was the same as the first part.2)The mononuclear cells of MLN,PPs,lamina propria of the small intestine(s LP)and lamina propria of the colon(c LP)were isolated.The proportion of Foxp3~+Tregs and IgA~+plasma cells in the mononuclear cells of each group was detected by flow cytometry.3)The feces of mice were collected and the level of IgA in feces was detected by ELISA method.The level of IgA in small intestine and colon was detected by immunohistochemistry.The proportion of IgA encapsulated bacteria was detected by flow cytometry.Results:1)Flow cytometry analysis showed that in PPs,compared with DSS+PBS group,the proportion of Foxp3~+Tregs in DSS+MSCs group was significantly increased(P<0.001),and the proportion of IgA~+plasma cells was also significantly increased(P<0.01).There was no significant difference between Control group and DSS+PBS group(all P>0.05).In s LP,the proportion of Foxp3~+Tregs and IgA~+plasma cells in DSS+MSCs group was significantly higher than that in DSS+PBS group(all P<0.05).In c LP,the proportion of Foxp3~+Tregs in DSS+MSCs group was significantly higher than that in DSS+PBS group(P<0.01),but the proportion of IgA~+plasma cells had no significant difference(P>0.05).Similarly,in MLN,the proportion of Foxp3~+Tregs in DSS+MSCs group was significantly higher than that in DSS+PBS group(P<0.01),but there was no significant difference in the proportion of IgA~+plasma cells(P>0.05).2)In small intestine,the expression of IgA in DSS+MSCs group was significantly higher than that in DSS+PBS group(P<0.05),but there was no significant difference in IgA expression between DSS+MSCs group and DSS+PBS group in colon(P>0.05).3)The results of ELISA showed that compared with the DSS+PBS group,the fecal IgA concentration in the DSS+MSCs group increased significantly on the 7th and 9th day(all P<0.001),but there was no significant difference on the 1st,3rd and 5th day between the two groups(all P>0.05).4)The results of bacterial flow showed that the proportion of IgA encapsulated bacteria in DSS+MSCs group was significantly higher than that in DSS+PBS group(P<0.05).Conclusions:1)HUMSCs promoted the plasma cell response of Foxp3~+Tregs and IgA~+in PPs and s LP with DSS-induced experimental colitis model mice.2)HUMSCs promoted the secretion of IgA and increased the prop ortion of IgA encapsulated bacteria in the intestinal tract of mice with e xperimental colitis induced by DSS. |
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