Microbial And Metabolomic Profiles Of Diarrhea-predominant IBS Patients And Their Correlation With Depression And Anxiety

Background and aimsIrritable bowel syndrome(IBS)is the most common functional bowel disease that features abdominal pain,bloating and altered bowel habits,which lasts for more than 3 months.Epidemiological studies have showed that IBS affected 7-21%of overallpopulation worldwide and 1-16%domestically.IBS is subdivided by stool character into several subtypes and diarrhea-predominant IBS(IBS-D)is the most common subtype domestically which features more than 25%of bowel movements indicated by the type 6/7 Bristol Stool Form Scale,and less than 25%of bowel movements type 1 or 2.Despite the relatively good prognosis,IBS affects the quality of life of patients immensely and extensively,and results in repetitive,long-term consultations and large medical costs.The current research suggests that IBS is a complex disease that fits the biopsychosocial-psycho-medical model with the involvement of multiple factors,such as diet,gut dynamics abnormalities,visceral hypersensitivity,abnormal brain-gut axis regulation,infection,immunity and other factors.As the main norm in the functional diseases in gastroenterology,the recently published Rome ? standard has defined functional gastrointestinal diseases,including IBS,as disorders in brain-gut interaction.Rome ? emphasizes the relationship between central processing and pathophysiology of functional gastrointestinal diseases.In addition to the above-mentioned gastrointestinal somatic symptoms,IBS is often accompanied by psychological symptoms such as anxiety and depression,altered behavioral patterns,impaired social functioning and personality abnormalities.Also,some studies have shown that psychosomatic symptoms in IBS patients are positively correlated with the severity of intestinal symptoms.Several studies have reported that IBS patients have significantly higher levels of anxiety,depression and behavioral abnormalities than healthy individuals.Recent research suggests that the intestinal microbiota is an important component of intestinal structure and function,involved in the physiological regulation and pathogenesis of the intestinal and systematic conditions.The gut flora regulates the central nervous system through multiple pathways,not only shaping the development of the nervous system,but also participating in the pathophysiological processes of a variety of mental and psychosomatic disorders,even influencing individual social behavior and cognitive function.Thus,the important concept of the microbiota-gut-brain axis has been formed as a complement and refinement to the gut-brain interaction.Existing evidences have identified intestinal dysbiosis in inflammatory bowel disease,major depressive disorder,autism spectrum disorder,etc.Recent high-throughput sequencing studies have shown that IBS patients may also have structural and functional disorders in gut microbiota associated with psychiatric symptoms such as anxiety and depression;Liu et al.found that the gut microbial structure of IBS patients is similar to that of patients with major depressive disorder,such as decreased diversity and increased abundance of Bacteroides and Prevotella.However,the conclusions of the current studies are still inconsistent and the specific gut bacterial components involved in the physical and mental symptoms of IBS patients are far from clear and require further investigation.On the other hand,with the development of metabolomics technology,researchers have been paying more attention to the role of metabolomic changes in pathophysiology of diseases.A growing number of studies confirm that small molecule metabolites may be an important pathway by which intestinal flora influence physiological activity,rendering them an important component of the microbiota-gut-brain axis.As the most complex metabolic reservoir in the body,the gut microbiota utilizes a variety of nutrients as substrates,forming numerous metabolites such as short chain fatty acids,amino acids,etc.Metabolic products that enter into the circulation may affect various physiological functions,including development of blood-brain barrier and central nervous system,emotional experience,pain perception and behavioral patterns.In recent years,there have been studies on metabolomic characteristics in animal IBS models and patients using bio-samples such as serum,feces and urine.In this study,we investigated specific bacterial components and differential metabolites associated with somatic symptoms and psychiatric symptoms(anxiety and depression)in patients with IBS.By establishing a correlation network of intestinal microbiota,differential metabolites and psychiatric symptoms,we hope to provide a complement to the microbiota-gut-brain axis and help diagnosis and treatment of IBS patients with anxiety and depression symptoms.MethodsPart 1 Association study of fecal microbiota with somatic and psychiatric symptoms in patients with IBS-DIBS-D patients were included according to the Rome ? criteria published in 2016.Meanwhile,healthy control(HC)subjects were recruited through advertisement and were defined as individuals with colonoscopy of no negative results and without current or previous gastroenterological symptoms nor history of chronic diseases.Demographic data were collected for enrolled IBS-D and HC subjects as well as clinical symptom evaluation based on the IBS symptom severity scale(IBS-SSS).Subjective and physician assessment of anxiety and depression levels in the IBS-D group was then conducted.We collected the fecal samples from IBS-D patients and HCs.V3-V4 high variable region of the 16S rRNA gene was amplified and sequenced in the fecal genome.Differences in fecal microbial structure and compositions between IBS-D and HC groups were then analyzed.Correlations between fecal microbial abundance and IBS clinical symptoms,anxiety/depression levels in patients were analyzed by Spearman rank correlation coefficient.Part 2 Correlation network of gut microbiota,differential metabolites and psychiatric symptoms in IBS-D patientsWe collected and stored urine samples from the population included in Part I.UHPLS-MS/MS method was used for the analysis of urine metabolomics.The denoised and trimmed metabolomic raw data were utilized for comparing the metabolomic structure between IBS-D and HC groups,and screening for differential urine metabolites.Associations between differential metabolites and clinical symptoms and intestinal flora abundance was then performed to construct a correlation network.ResultsPart 11.Characteristics of subjectsA total of 70 IBS-D patients and 46 healthy controls were included.No significant differences in demographic data between 2 groups were detected and the Bristol stool form scale was significantly higher in the IBS-D group than in healthy controls(P<0.001).For IBS symptom severity scores,the IBS-D patient group was significantly higher than the control group(P<0.001).Scale assessment of anxiety and depression levels in IBS-D patients scored 49.89 ± 9.59 on the SDS,48.81± 9.79 on the SAS,16.57 ± 6.58 on the HADS,13.23± 5.15 on the HAM-D and 14.53 ± 5.86 on the HAM-A.2.Diversity analysis of fecal microbiota in IBS-D group and healthy controls Eligible fecal samples were collected from 70 IBS-D patients and 44 healthy controls.An average of 52298 sequences were obtained per sample.Simpson index was significantly higher in the IBS-D group(P=0.041),indicating relatively higher biodiversity,while the Ace index(P=0.007)and Chao index(P=0.018)were both lower than those in the control group,reflecting lower richness in 1 BS-D fecal microbiota3.Comparison of inter-group microbial community structure and species compositionHierarchical clustering and principal co-ordinates analysis(PCoA)based on bray-curtis distances showed that the community structure was similar between the IBS-D and HC samples.The relative abundance of Firmicutes was significantly lower in IBS-D patients(adjusted P<0.05),whereas there was no significant difference in Bacteroidetes(adjusted P>0.05)or Proteobacteria(adjusted P>0.05)between groups.At the genus level,Prevolella(adjusted P=0.041)was relatively more abundant in the IBS-D group,while Faecalibacterium(adjusted P<0.05),Eubacterium rectale group(adjusted P<0.05)and Subdoligranulum(adjusted P<0.05)were more abundant in feces from HCs.No significant divergences in Bacteroides(adjusted P=0.826)and Blautia(adjusted P=0.137)were found,which were the most abundant genera of IBS-D and HC group,respectively.4.Association analysis between fecal microbiota and anxiety and depression levels in IBS-D patientsBased on relative abundance at the genus level,there were significant negative correlations between Dialister and IBS symptom severity(r=-0.285,P=0.017),anxiety level(r=-0.347,P=0.003),and depression level(r=-0.308,P=0.010).Roseburia was also significantly negatively associated with IBS symptom severity(r=-0.298,P=0.012).In the multivariate analysis for which gender,age and IBS severity were adjusted,negative correlations were found between the relative abundance of Dialister and self-reported anxiety and depression levels,and Clostridium sensu stricto and self-reported anxiety and depression levels.Part 2Eligible urine samples were collected from 70 IBS-D patients and 42 healthy controls for the UHPLS-MS/MS process.After validation of metabolomics data by orthogonal partial least squares-discrimination analysis(OPLS-DA),114 significant differential metabolites between IBS-D and healthy controls were detected and 50 differential metabolites were annotated in the KEGG database.A total of 20 annotated differential metabolites were associated with anxiety and depression levels in IBS-D patients,including 3,4-dihydroxymandelaldehyde with SAS scores(r=-0.383,P 0.001),1-methylxanthine with SDS scores(r=-0.347,P=0.004),4-hydroxyphenylacetylglycine with SAS scores(r=-0.337,P=0.005),and 1 D-chiro-inositol with SAS scores(r=-0.336,P=0.005),etc.Combining the data of fecal microbial abundances,3,4-dihydroxymandelaldehyde(r=-0.293,P=0.014)and 1-methylxanthine(r=-0.299,P=0.012)levels were also negatively correlated with Clostridium sensu stricto abundance in IBS-D patients.ConclusionsThe fecal microbiota of IBS-D patients showed higher diversity and lower richness compared with healthy controls,but the overall community structures of 2 groups were not divergent as reflected by ? diversity.In IBS-D fecal bacteria,the abundance of Prevotella was elevated,while the abundance of Faecalibacterium,Eubacterium rectale group and Subdoligranulum was decreased.Relative abundances of Dialister,Roseburia and Clostridium sensu stricto were negatively correlated with anxiety and depression levels in 1 BS-D patients.There were differences in urine metabolomic characteristics of IBS-D patients and healthy controls,and levels of multiple differential metabolites were correlated with anxiety and depression levels in patients.Among them,urinary differential metabolites such as 3,4-dihydroxymandelaldehyde and 1-methylxanthine were negatively associated with Clostridium sensu stricto abundance.The study suggests that there is a complex regulatory network between psychiatric symptoms,intestinal flora and metabolomic changes in IBS-D patients,and that some of the differential bacteria and metabolites may have the potential as targets for the evaluation and treatment of anxiety and depression co-morbidity in IBS-D patients.