| Part One Clinical characteristics and intestinal microbiotas of patients with IBS-DObjective:To evaluate the demographics,symptom spectrum,quality of life,and psychological status in patients with IBS-D patients by using a series of questionnaires.And to compare and analyze the microbiological characteristics between fecal flora and mucosal flora in patients with IBS-D.Methods:From January 2022 to May 2022,37 IBS-D patients who met the Rome Ⅳ diagnostic criteria in the outpatient department of gastroenterology were continuously enrolled in the experimental group.Fifteen healthy subjects without organic intestinal diseases or gastrointestinal symptoms were recruited as the control group.All subjects were required to meet the admission criteria:The age was between 18 and 65 years old;the liver and kidney function was normal;the thyroid function was normal.Exclusion criteria:major history of gastrointestinal surgery;history of malignant tumor;pregnant and lactating women;patients with mental illness.All the subjects were investigated by questionnaire.The stool and mucosa biopsy samples of10 patients with IBS-D were collected.The contents of the questionnaire include general clinical demographic characteristics,IBS-SSS,IBS-QOL,GSRS,HADS and PSQI.Fecal and sigmoid mucosa biopsies were used for intestinal flora detection and species analysis by 16SRNA high-throughput sequencing.Compare the species characteristics and differences of fecal flora and mucosal flora in patients with IBS-D,and evaluate whether the fecal samples can express the true situation of intestinal flora in the study of colonic flora of patients with IBS-D.Results:1.The average age of 37 patients(28 males and 9 females)with IBS-D was 39.27±12.04years,and the average BMI was 23.88±3.41kg/m~2.2.There were 37 patients in IBS-D group,the IBS-SSS symptom score was 289.19±64.57.Among them,70.27%(26 cases)were moderate symptoms and 29.73%(11 cases)were severe patients.3.The scores of GSRS,HADS and PSQI in IBS-D group were significantly higher than those in HC group(P<0.05).4.At the OTU level,a total of 1360 OTU were detected in the feces and mucosa of IBS-D patients,of which 396 OTU were detected in the two groups.There were 931 OTU specific to mucosal samples and only33 OTU specific to fecal samples.5.Ace index and Chao1 index in stool group of IBS-D patients were significantly lower than those in mucous membrane group,and Shannon and Simpson index in stool group were also lower than those in mucous membrane group,but there was no significant difference.6.The results of Beta diversity analysis showed that there were significant differences in fecal and mucosal flora in patients with IBS-D.7.There were great differences in species structure between fecal flora and mucosal flora in patients with IBS-D.Conclusions:1.Patients with IBS-D have prominent gastrointestinal symptoms,mental and psychological problems and sleep disorders.2.The diversity of mucosal flora in patients with IBS-D is more abundant than that in feces.3.There are significant differences in microbiota structure between mucous membrane and feces of patients with IBS-D.Mucosa microbiotas may provide more accurate and rich microbial species information for exploring the pathogenesis of IBS-D.Part Two Analysis of colonic mucosal microbiology and metabonomics in patients with IBS-DObjective:To compare the differences of colonic mucosal microbiology and mucosal metabonomics between IBS-D and healthy subjects.To screen out differential flora and differential metabolites in the colonic mucosa and to explore their role in the pathogenesis of IBS-D.Methods:From June 2022 to July 2022,10 IBS-D patients who met the Rome Ⅳ criteria in the outpatient department of gastroenterology were continuously enrolled as the experimental group(IBS-D group).Six healthy subjects without organic intestinal diseases and gastrointestinal symptoms were recruited as control group(HC group).Two biopsies of ileocecal mucosa were taken from all subjects during enteroscopy.One mucosal sample was sequenced by 16Sr RNA high-throughput sequencing,and the species structure of intestinal mucosal flora was analyzed.The other mucosal mucosa was analyzed by DESI-MSI.To analyze and compare the microbial structure and metabolic spectrum of colonic mucosa in IBS-D group and HC group.Search for differential microflora and differential metabolites,and use KEGG to analyze the metabolic pathway that may be involved in the pathogenesis of IBS.Results:1.Chao1 index in IBS-D group was significantly higher than that in HC group,while Richness and Simpson index in IBS-D group was also higher than that in HC group,but there was no significant difference.Shannon,Dominance and Equitablity index in HC group was higher than that in IBS-D group,but there was no statistical difference.2.Through the Beta diversity analysis of intestinal mucosal flora,it was found that the difference between IBS-D group and HC group was greater than that within group,and the clustering degree of HC group was better than that of IBS-D group.3.Species difference analysis showed that compared with HC group,Faecalimonas,Gp3,Ligilactobacillus,Acinetobacter,Megasphaera,Shuttleworthia,Akkermansia,Cetobacterium,Veillonella,Collinsella,Bradyrhizobium and Alteromonas in colonic mucosal flora of IBS-D group increased,while Phocaeicola and Barnesiella decreased.4.A total of 2930 metabolites were detected in colonic mucosa of IBS-D group and HC group in positive ion mode and 1456 in negative ion mode.5.Compared with HC group,there were 141 upregulated metabolites(123 in positive ion mode and 18 in negative ion mode)and904 down-regulated metabolites in IBS-D group(196 in positive ion mode and 708 in negative ion mode).Among them,there were 41 lipids differential metabolites,7 up-regulated lipids and 34 down-regulated lipids.6.KEGG Enrichment analysis of differential metabolic lipids in intestinal mucosa of IBS-D showed that compared with HC group,the expression of seven medium-chain or long-chain fatty acids Palmitic acid,Arachidic acid,Oleic acid,Linoleic acid,Alpha-Linolenic acid,Myristic acid and Dodecanoic acid were down-regulated,of which Oleic acid,Linoleic acid and Alpha-Linolenic acid were unsaturated fatty acids.7.There was a down-regulation of unsaturated fatty acid biosynthesis in colonic mucosa of patients with IBS-D.Conclusions:1.Patients with IBS-D have dysbiosis of mucosal flora and disorders of mucosal metabolism.2.The occurrence of IBS-D can be mediated by an increase in the number and species of harmful intestinal bacteria and a decrease in the number and species of beneficial bacteria.3.Medium and long chain fatty acids may be potential biomarkers of IBS-D,and down-regulation of the expression of their related metabolic pathways may be involved in the pathogenesis of IBS.Part Three Research on Mucosal flora and mucosal metabolomics in different parts of colonObjective:To compare and analyze the structure of mucosal microbial species and mucosal metabolites in different parts of the colon of IBS-D patients and healthy subjects separately.Methods:From July 2022 to August 2022,10 IBS-D patients who met the Rome Ⅳ diagnostic criteria in the outpatient department of gastroenterology were continuously enrolled in the IBS-D group,and 6healthy people were recruited into the HC group.Two mucous membranes of ileocecal colon and sigmoid colon were taken from all subjects during enteroscopy.One mucosal sample was sequenced by16Sr RNA high-throughput detection,and the composition of microbial species was analyzed.The other was detected and analyzed by DESI-MSI.To compare the microflora structure and mucosal metabolic spectrum of ileocecal mucosa and sigmoid mucosa in patients with IBS-D and healthy people(HC group).Results:1.In patients with IBS-D,the Richness,Chao1 and Simpson index of ileocecal mucosa was slightly higher than that of sigmoid mucosa,while the Shannon,Dominance and Equitablity index of ileocecal mucosa was slightly lower than that of sigmoid,but the difference was not statistically significant.PCo A analysis showed that the clustering degree of mucosal microbial structure in the two parts of colon was similar.2.In HC group,the Simpson index of ileocecal mucosa was slightly higher than that of sigmoid mucosa,while the Richness index,Chao1,Shannon,Dominance and Equitablity index of ileocecal mucosa were slightly lower than those of sigmoid mucosa,but there was no significant difference.PCo A analysis showed that the clustering degree of microbial species in the two colonic mucosa was also similar.3.Multivariate statistical analysis showed that the metabolic profiles of colonic mucosa in both IBS-D group and HC group were similar.Conclusions:The structure of mucosal flora and metabolic profiles in different parts of colon in patients with IBS or healthy people is similar. |
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