| Part 1 Study on the distribution characteristics of gut microbiota and its correlation with clinical symptoms in patients with irritable bowel syndromeObjective:In this study,16 S rDNA sequencing technology was used to study the differences in the structure and species composition of gut microbiota between patients with irritable bowel syndrome(IBS)and healthy volunteers in Lanzhou.Analyze the correlation between intestinal flora and clinical symptoms,and preliminarily explore its possible mechanism,so as to provide basis for accurate diagnosis and treatment.Materials and Methods:According to the Rome IV criteria,33 IBS patients who met the inclusion criteria of this experiment who were treated in the outpatient department of Lanzhou University Second Hospital from June 2019 to November 2019 were prospectively collected as the IBS patient group.31 healthy volunteers served as the health control(HC)group;fresh stool samples were collected from all subjects,and the basic information of the subjects and the clinical phenotype score data of the patients were collected.The 16 S r DNA amplicon sequencing technology was used to perform V3-V4 variable region amplification sequencing on the stool samples of all subjects,and the differences in the intestinal flora diversity and microbial species composition between IBS patients and HCs were analyzed.Spearman correlation analysis was used to calculate the correlation between different bacteria and various clinical phenotypic evaluation results.Results:1.IBS symptom severity scale(IBS-SSS),visceral sensitivity index(VSI),Pain Catastrophizing Scale(PCS),Hamilton anxiety scale(HAMA)and Hamilton Depression Scale(HAMD)scores in the IBS group were significantly higher than those in HC group(P < 0.01);IBS quality of life scale(IBS-QOL)scores showed the average score of IBS group Significantly lower than the HC group(P<0.01).2.Compared with HCs,there was no significant change in fecal microbial alpha diversity in IBS patients in Lanzhou area.Beta diversity analysis found that the differences in the composition of intestinal fecal samples between the IBS group and the HC group were larger than the differences within the groups,but the overall analysis found that the composition of the samples in this experiment had a high similarity,and it was difficult to identify different groups in the overall colony characteristics.3.The rank sum test showed a total of 15 different species at all species taxonomic levels between the two groups,and the increase in the genus Dorea in the IBS group was significantly positively correlated not only with the IBS-SSS score of IBS patients,but also with the IBS patients‘ health patient questionnaire,Simplified Mc Gill Pain Scale,PCS,HAMD,HAMA.Elevated Clostridium XVIII abundance was positively correlated with HAMD scores in patients.Conclusion:There were differences in fecal microbial species composition between IBS group and HC group,and the abundance of different species was correlated with clinical symptoms,anxiety and depression of patients.Part 2 Biomarker research and mechanism exploration in patients with irritable bowel syndrome based on non-target metabolomicsObjective:High-resolution non-targeted metabolomics technology was used to detect metabolites in plasma of IBS patients and HCs,and the differential metabolites were screened and the biomarkers with clinical diagnostic value were selected.At the same time,taking the differential metabolites as clues,the key metabolic pathways were annotated with the help of bioinformatics analysis to explore the key metabolites and pathways related to the pathogenesis of IBS.Materials and Methods:Subjects were included in the same section as the previous section,and 64 collected plasma samples(33 samples from IBS patients)were analyzed by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry for off-target metabolomics analysis.Metabolite identification statistics were performed on the obtained detailed plasma metabolic characteristics information,and multi-dimensional statistical analysis combining univariate and multivariate was used to screen differential metabolites and mine biomarkers.At the same time,bioinformatics analysis of differential metabolites was performed to annotate related metabolic pathways.Finally,the correlation analysis between the differential metabolites and the previous part of the intestinal flora and clinical symptom phenotypes was carried out.Results:1.A total of 271 metabolites were identified in this project,including 168 in positive ion mode and 144 in negative ion mode,of which 41 were overlapping metabolites in the two modes.Through multidimensional statistical analysis,with VIP>1 and P<0.05 as the screening criteria for significantly different metabolites,a total of 17 significantly different metabolites were obtained.Using receiver operating characteristic(ROC)curve to evaluate the predictive performance of differential metabolites(the ability to distinguish between IBS group and HC group),it was found that Norethindrone Acetate had the highest predictive value,and the area under the curve(AUC)was 0.7863,followed by the predicted value of Tyramine with an AUC of 0.7671.2.Through the analysis of KEGG-enriched gene pathways,it was found that protein digestion and absorption,cancer-related central metabolism,mineral absorption,and amino acid biosynthesis were the metabolic and signal transduction pathways that were significantly affected in IBS.3.The correlation analysis of differential metabolites and clinical phenotypes found that Pinocembrin was positively correlated with disease course,N1-Methyl-2-pyridone-5-carboxamide was positively correlated with anxiety and depression scores,and L-Valine was positively correlated with VSI and PCS.Conclusion:1.There are disorders in blood metabolism in PATIENTS with IBS,and the differential metabolites are expected to serve as potential biomarkers to identify patients with IBS and HCs.2.IBS plasma differential metabolites were associated with clinical course,anxiety,depression and visceral sensitivity of patients.Part 3 Study of gut microbiota-related metabolites in patients with irritable bowel syndrome based on targeted metabolomicsObjective:High-resolution targeted metabolomics technology was used to quantitatively detect metabolites such as short chain fatty acids(SCFA),tryptophan,and neurotransmitters that are closely related to gut microbiota in plasma from IBS patients and HCs.Summarize the changes of specific metabolites in plasma of IBS patients,study the relationship between intestinal flora and metabolites,explore the relationship between metabolic abnormalities and clinical symptoms and phenotypes,and provide an objective basis for accurate diagnosis,research and development of treatment targets and pathogenesis of the disease.Materials and Methods:Based on the gas chromatography-mass spectrometry platform,the multiple reaction monitoring technology was used for the collected 64 plasma samples,and the standard substance was used as a reference to conduct targeted and specific analysis of SCFAs,tryptophan and neurotransmitters in plasma,and the absolute quantitative value in plasma were obtained.At the same time,the quantitative data of the obtained metabolites were statistically analyzed to screen the differential metabolites.Finally,the correlation analysis between the screened differential metabolites and the intestinal flora and clinical symptom phenotype data in the previous part of the research results was carried out.Results:1.The quantitative results of SCFAs showed that there was no significant difference in plasma total SCFA,acetic acid,propionic acid,isobutyric acid,valeric acid and caproic acid between IBS group and HC group,but there were significant differences in butyric acid and isovaleric acid content,butyric acid content in IBS group was significantly lower than that in the HC group(P<0.05),and the isovaleric acid content was significantly higher than that in the HC group(P<0.05).2.The levels of 5-hydroxy-L-tryptophan,kynurenine and N-formylkynurenine in the IBS group were significantly lower than those in the HC group(P<0.01).The levels of 3-lactic acid,indole-3-carbaldehyde,3-methyl indole,tryptophan,3-hydroxy-L-kynurenine and cinnabaric acid were significantly higher than those in the HC group(P<0.01).3.IBS significantly changed the composition of the overall neurotransmitters in plasma,among which the contents of aspartic acid,glutamic acid and spermine were significantly higher than those in the HC group(P<0.05).The contents of3,4-dihydroxyphenylacetic acid,cysteine and norepinephrine in the HC group were significantly higher than those in the IBS group(P<0.05).4.5-hydroxy-L-tryptophan was negatively correlated with the abundance of Leuconostocaceae.Cysteine was negatively correlated with the abundance of Oxalobacteraceae.Clostridium XVIII,and the Valeric acid content was negatively correlated with Porphyromonadaceae abundance.The content of tryptophan was positively correlated with the abundance of Desulfovibrio,the content of spermidine and aspartate was positively correlated with the abundance of Dorea,and the content of 5-hydroxy-L-tryptophan was positively correlated with the abundance of Weissella.5.Butyric acid was negatively correlated with VSI score,3,4-dihydroxyphenylacetic acid was positively correlated with VSI,aspartic acid was positively correlated with IBS-SSS score,glutamate was positively correlated with HAMA and HAMD scores,3-hydroxy-L-kynurenine was significantly positively correlated with HAMD scores,and N-formyl-kynurenine and norepinephrine were negatively correlated with disease course.Conclusion:1.Abnormal plasma metabolism in patients with IBS mainly comes from intestinal microbiota related metabolism,which may be related to intestinal microbiota disorder in patients with IBS.2.The plasma levels of butyric acid in PATIENTS with IBS are significantly reduced and isovaleric acid is significantly increased,which in turn upregulates key neurotransmitters such as tryptophan,glutamate and metabolites,and regulates emotions such as depression and anxiety through the enteric-brain axis,thus causing psychiatric symptoms.3.There was a negative correlation between plasma butyric acid and intestinal sensitivity in IBS patients,and the decrease of butyric acid content exacerbated intestinal symptoms.Part 4 Functional magnetic resonance imaging-based study of cerebral hemisphere diencephalic connectivity in patients with irritable bowel syndromeObjective:Hemispheric asymmetry and interhemispheric connectivity in IBS patients were assessed by multimodal functional magnetic resonance imaging(f MRI).At the same time,the correlation between brain imaging features and clinical symptom phenotype scores,plasma key metabolites,and important intestinal flora of IBS patients was analyzed to explore the possible mechanism of abnormal gut-brain interaction in IBS patients.Materials and Methods:The subjects were included is the same as the first part.A total of 34 IBS patients and 33 HCs underwent multimodal f MRI in this study.The data acquisition included structural MRI,resting-state f MRI and diffusion tensor imaging.Calculate and evaluate the differences of voxel-mirrored homotopic connectivity(VMHC),fractional anisotropy(FA),fiber length(FL),fiber number(FN),and asymmetry index(AI)between groups.At the same time,the correlation analysis of differential brain imaging indexes and clinical symptom phenotype scores,plasma metabolomics differential metabolites,and important intestinal flora of IBS patients was carried out,and a comprehensive co-occurrence network was constructed based on the correlation results.Results:1.VMHC between groups showed that VMHC in superior occipital gyrus,middle occipital gyrus,precuneus,posterior cingulate gyrus and angular gyrus in IBS patients were higher than those in HC group.VMHC in auxiliary motor area of IBS patients was lower than that of HCs(P < 0.05,Alphasim correction).2.The anatomical connection between groups showed that AI,FA,FL and FN had no significant difference between IBS group and HC group.Pearson correlation analysis showed that FA(r =-0.34,P < 0.05)and FL(r =-0.4,P < 0.05)in the posterior part of bilateral cingulate gyrus were significantly negatively correlated with the severity of IBS.Compared with HC group,the level of interhemispheric functional connection in bilateral superior occipital gyrus,middle occipital gyrus,precuneus,cingulate gyrus and angular gyrus in IBS group was higher,while the level of auxiliary motor area was lower.3.Based on the correlation between brain imaging indexes and clinical phenotype score,plasma metabolite level and relative abundance of intestinal flora,a comprehensive co-occurrence network was constructed.It was found that compared with HC group,the overall network connection level of IBS group decreased,especially the connection between metabolites decreased,and there were new connections between flora and metabolites,metabolites and clinical phenotype.Conclusion:1.The functional connections between the cerebral hemispheres in IBS patients are more susceptible to the disease itself than the anatomical connections.The affected areas are mainly concentrated in the default network and the sensorimotor network,and the brain has the most stable structural morphology and is not easily affected by the disease.2.In the co-occurrence network constructed based on the multi-omics correlation results,the co-occurrence network of the IBS group was abnormal.3.FMRI can be used as an effective bridge to study the abnormal gut-brain interaction in IBS,which can help to reveal the pathophysiological mechanism of IBS from the system biology level by combining multi-omics. |
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