| Background: The depression in patients with inflammatory bowel diseases(IBD)is prevalent.There is a close correlation between depression and IBD.Research has shown that depression is a risk factor for the onset and progression of IBD.At the same time,IBD can also induce the occurrence of depression,so depression and IBD are extremely prone to form a vicious cycle.Research has found that compared with patients with IBD,patients with depression(IBDD)are more seriously ill,have a heavier disease burden,and have poor clinical efficacy and poor prognosis.Intestinal microflora is closely related to the field of IBD and mental diseases such as depression.Supplemental beneficial bacteria therapy has been a research hotspot in recent years,but the characteristics of intestinal microflora in IBDD are unknown.This study is the first to explore the characteristics of intestinal microflora in patients with IBD and depression,in order to identify beneficial bacteria that can alleviate colitis and improve depression,which has important potential clinical value for the treatment of IBDD patients.Method:(1)In order to investigate the depression status of IBD patients and the relationship between depression and other factors(anxiety,sleep,quality of life,disease activity),basic information was collected from 117 IBD patients(including 86 CD patients and 31 UC patients)and 68 healthy controls.The patient health survey questionnaire PHQ-9,generalized anxiety questionnaire GAD-7,Pittsburgh sleep quality index PSQI,and inflammatory bowel disease quality of life questionnaire IBDQ were used for analysis,the relationship between PHQ-9 score and disease activity,and the correlation between patients’ depression level and anxiety,sleep,and quality of life were analyzed.(2)In order to screen the key bacteria in IBDD,fecal metagenomics was used to analyze the feces of 6 CD patients with depression(CDD)and16 CD patients without depression(CDND).And the result was validated in an expanded population through q PCR: including 25 CDD,24 CDND,10 UC patients with depression(UCD),and 5 UC patients without depression(UCND).Simultaneously the result was validated in the human gut microbiome metagenomic database GMrepo.(3)A mouse model of colitis induced by sodium dextran sulfate(DSS)and trinitrobenzene sulfonic acid(TNBS)was used to investigate whether the bacterium has the effect of alleviating colitis.At the same time,differential gene analysis and functional enrichment analysis were conducted through RNA sequencing in intestinal tissue to preliminarily explore the molecules and pathways affected by it,and partial differential genes were verified using q PCR.(4)To construct DSS colitis mice and determine whether DSS mice develop depression through animal behavioral analysis(open field test and tail suspension test).In DSS colitis model and the intraperitoneal injection of LPS depression mouse model,the selected key differential bacteria were administered orally for intervention,and animal behavioral analysis was conducted to explore whether the bacteria had the effect of alleviating depression in mice.Brain derived neurotrophic factor(BDNF),a depression related substance,was detected in the hippocampus of mice.(5)HM350 metabolomics was used to analyze the feces and culture supernatant of DSS colitis mice with this bacterium intervention to find the key metabolites related to this bacterium.Intervention of the DSS colitis mouse model with the above key metabolites to explore whether it has the effect of alleviating colitis.Intervention with the above key metabolites in DSS colitis mice and intraperitoneal injection of LPS depression mouse models to explore whether it has the effect of improving depression.(6)HM350 metabolic group was used to analyze the metabolites of peripheral blood and brain tissue in DSS colitis mice treated with this bacterium,and the metabolites of intestinal tract,peripheral blood and brain tissue were analyzed to explore whether they had common and differential metabolites.Methods such as Evans blue(EB)blood brain barrier permeability test,electron microscopic analysis of the mouse blood brain barrier,and murine blood brain barrier tight junction protein Claudin-5 immunofluorescence were used to investigate whether the bacterium and its metabolites can regulate the integrity of the blood brain barrier at the animal level;Transmembrane electrical resistance(TEER)measurement and expression of mouse cerebral vascular endothelial cell tight junction protein Claudin-5 were used to determine whether this metabolite can regulate the integrity of the blood brain barrier destroyed by LPS in mouse cerebral vascular endothelial cells.Results:(1)The PHQ-9,GAD-7,and PSQI scores of CD and UC active stage patients were higher than those of remission stage patients and healthy controls;The IBDQ score of patients in the active phase is lower than that of patients in the remission phase.PHQ-9 has good accuracy in distinguishing between active and remission stages of patients.Correlation analysis found a significant correlation between PHQ-9 scores and GAD-7,PSQI,and IBDQ scores in CD and UC patients.(2)The fecal metagenomic analysis of 6 CDD and 16 CDND patients showed that the bacterial abundance of Bacteroides vulgatus(B.vulgatus)in CDD was significantly lower than that in CDND.Using q PCR to verify the result in more patients,the abundance of B.vulgatus was lower in CDD and UCD.At the same time,it was found that the abundance of B.vulgatus was lower than that of healthy controls.In addition,in the public database GMrepo,comparing IBD with healthy people,depression with healthy people,it was found that the abundance of B.vulgatus is lower in IBD and depressive populations,suggesting that B.vulgatus is closely associated with IBD and depression.(3)The administration of B.vulgatus can significantly improve weight loss,colon shortening,disease activity index,and intestinal inflammatory cell infiltration in DSS and TNBS induced-colitis mice.Differential gene analysis,gene function enrichment analysis,and q PCR validation after RNA sequencing in colon tissue showed that B.vulgatus may alleviate colitis by inhibiting the activation of the IL-17 signaling pathway like reducing the expression of molecules in this pathway such as S100a8,S100a9,IL-6,CXCL1,and CXCL2.(4)Using open field test and tail suspension test,it was found that the mouse activity distance decreased and the tail suspension immobility time increased,indicating that DSS mice have significant depressive behavior.In addition,immunofluorescence suggests that the expression of depression related molecule BDNF in the hippocampus of DSS mice is significantly downregulated.The administration of B.vulgatus can significantly improve the depressive behavior,and reverse BDNF reduction.Meanwhile,in a mouse model of LPS induced depression,The administration of B.vulgatus and the antidepressant drug fluoxetine have the same improvement effect on depressive behavior in mice.(5)The metabolite of intestinal flora is the key substance for its function,in order to clarify the possible ways in which B.vulgatus can exert anti-inflammatory and antidepressant effects,the fecal metabolisms of DSS mice and culture medium treated with B.vulgatus are analyzed.4-hydroxyphenylacetic acid(4-HPAA),chenodeoxycholic acid(CDCA),3-aminosalicylic acid(3-ASA),4-hydroxyproline(4-HP)were detected as the four metabolites related to B.vulgatus.It was found that 4-HPAA,CDCA,and 3-ASA of the above four metabolites can alleviate weight loss,shortened colon length,disease activity index,and inflammatory degree of colon tissue pathology in colitis mice.However,4-HP has no significant relief effect on colitis.Among the above three metabolites for relieving colitis,only 4-HPAA has the effect of improving depressive behavior in DSS colitis and LPS models,suggesting that 4-HPAA may be a key metabolite for B.vulgatus to play an anti-inflammatory and antidepressant role.(6)The gut-brain axis is an information network that connects the intestine and the brain.The metabolite related to intestinal bacteria enter the blood and brain is an important pathway for the gut-brain axis.To investigate whether 4-HPAA plays a role through the gut-brain axis,we analyzed the metabolomics of mouse peripheral blood and brain tissue,and found that 4-HPAA was significantly upregulated in the peripheral blood of mice,but there was no significant change in brain tissue.Since 4-HPAA does not enter the brain,and the destruction of the blood-brain barrier is one of the important mechanisms of depression,we further investigate whether 4-HPAA affects the integrity of the blood-brain barrier.The EB permeability experiment found that the permeability of the blood brain barrier in DSS and LPS mice was significantly increased,while the intervention of B.vulgatus and 4-HPAA could resist the increase in the permeability of the blood brain barrier.The results of brain tissue electron microscopy suggest that the intervention of B.vulgatus and 4-HPAA can improve the changes of blood brain barrier related components in DSS mice,such as basement membrane rupture,expansion of perivascular space,damage of tight junctions,and swelling of the distal feet of astrocytes.At the same time,brain tissue immunofluorescence found that the expression level of the important tight junction protein Claudin-5 in the blood brain barrier of DSS mice was significantly reduced,B.vulgatus and 4-HPAA can upregulate the expression of Claudin-5.Transmembrane electrical resistance(TEER)of cerebral vascular endothelial cells treated with LPS decreased,and the protein expression of Claudin-5 decreased.Intervention with 4-HPAA can reverse these changes.The above results suggest that the permeability of the blood brain barrier in depressive mice is increased,and 4-HPAA has the effect of enhancing the integrity of the blood brain barrier.Conclusion:(1)Patients with active IBD have a high level of depression,which is closely related to anxiety,sleep,and quality of life;(2)Bacteroides vulgatus is the most prominent bacterial species in the feces of IBD patients with depression,and it has the effect of alleviating intestinal inflammation and depression;(3)Bacteroides vulgatus may alleviate intestinal inflammation by downregulating the expression of IL-17 signaling pathway related factors,while its key metabolites 4-HPAA,CDCA,and 3-ASA can improve intestinal inflammation in mice;(4)The antidepressant effect of Bacteroides vulgatus is related to its key metabolite4-HPAA regulating the integrity of the blood-brain barrier. |
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