16S-rRNA Gene Sequencing Study Of Intestinal Microbiome Of Inflammatory Bowel Disease And The Effect Of Probiotic On Enteritis At Different Stages

Background and AimsInflammatory bowel disease(IBD)includes ulcerative colitis(UC)and Crohn disease(CD).It is a recurrent intestinal inflammatory disease with unknown pathogenesis.Recent studies have shown that the cause may be spontaneous inflammation of multiple factors,such as intestinal immunity to microorganisms.The method of 16S-rRNA gene sequencing can reveal the characteristics of intestinal flora in patients with UC.However,the correlation between intestinal microbial changes and disease progression is still unclear.The course of UC disease is related to the assessment of the severity of the disease,having a serious negative impact on the quality of patients' life,and even increase the risk of colorectal cancer.The intestine requires a complex and highly regulated intestinal immune response to achieve a balance between immune tolerance and immune response.Studies have shown that UC is accompanied by a variety of immune cell dysfunction and related cytokine regulation imbalance.Dendritic cells(DCs)are present in most tissues of the body,recognizing antigens,and play a pivotal role as an immune hub in the differentiation of T cells.Immune checkpoint molecules expressed on the surface of immune cells,such as T cell immunoglobulin and mucin-domain containing molecule 3(Tim3),participating in the regulation of the local microenvironment and are regulators of immune tolerance.However,Tim3's mechanism of action on DC in different stages of enteritis is rarely reported.Probiotics have been used as adjuvant therapy in UC,Lactobacillus reuteri(L reuteri)and Clostridium butyricum(C.butyricum)can inhibit the production of pro-inflammatory cytokines.However,in different processes of enteritis,the regulation of probiotics on innate immunity is rarely reported.We hypothesized that there may be some special intestinal flora characteristics while disease development,and it has a guiding role in the intervention treatment of probiotics.To test this hypothesis,we investigated the changes in the intestinal flora of healthy volunteers and UC patients,as well as the mechanisms by which probiotics regulate the innate immunity of mice at different stages of enteritisMethodPart 11.The general view of stool samples from patients and healthy controlsRecruit clinical UC patients and healthy volunteers to collect feces,and analyze the diversity of microbiota and structural composition of fecal bacteria in IBD patients based on the 16S-rDNA high-throughput sequencing technology.2.The impact of disease progression on intestinal floraUC patients are divided into short-term disease course(SC:disease course is 1-2 years),medium-term disease course(MC:disease course is 3-9 years)and long-term disease course(LC:disease course is greater than or equal to 10 years)three categories Investigate whether the fecal flora diversity and bacterial community composition are differentPart 21.The innate immune status of acute enteritis and the influence of probiotic interventionTo study the changes of the proportion and subgroups of intestinal DC and the expression difference of Tim3 in DSS acute enteritis model mice by Lactobacillus reuteri probiotic intervention.2.The innate immune effects of probiotics and the metabolites on low-grade enteritisUsing TNBS model mice with low-grade enteritis,intervention with C.butyricum and its metabolite butyrate to study changes in intestinal DC ratio and subgroups.Figure the differences in expression of Tim3 and compare with acute enteritis ResultsPart 11 The diversity of UC fecal flora is increased.Compared with the HC group,the levels of Proteobacteria and Actinobacteria decreased in the UC group,while Firmicutes increased significantly.2 The number of Firmicutes bacteria in patients with a disease course of more than 10 years has not decreased significantly.Bifidobacterium,Blautia only changed significantly in the early stages of the disease.Enterococcus are also significantly higher in patients with a long course of disease than short-term diseases and healthy controls,Part 21.The expression of CD 11 c+CD 11 b+DCs and CD 11 c+103+DCs in the colon of DSS-induced colitis mice was significantly lower than that of normal control mice(P<0.05)In addition,the intervention of L.reuteri promoted the proportion of CD11c+CD11b+DCs and CD11c+103+DCs.These results indicate that L.reuteri can activate the protective effects of tolerant DCs,and reduce DSS-induced intestinal inflammatory immune responses2.TNBS low-grade inflammatory pathology has no obvious changes,but inflammatory cytokines still increase.C.butyricum and butyrate can reduce the expression of IL-17A,and also significantly increased the spleen CD11c+CD11b+DCs IL-10 secretion.SignificanceThis study combine changes in the intestinal flora of UC with the duration of the disease,which may help guide the staging of UC.Explore the effects of probiotic interventions on the intestinal innate immune status in different disease periods.The disadvantage of this subject is that the specific action of probiotics on DCs and the impact on the intestinal flora after intervention still need to be further explored.