Oral Probiotics On Anxiety-and Depressive-like Behaviors Induced By Chronic Mild Stress In Mice And The Effects Of Gut Microbiota-inflammation-brain Axis

Objective:In this study,we explored the potential anti-anxiety and depression effects of multi-strain probiotics(Lactobacillus helveticus R0052+Lactobacillus plantarum R1012+Bifidobacterium longum R0175)and its effects on gut microbiota,inflammatory cytokines in the serum and hippocampus,and indoleamine 2,3-dioxygenase-1(IDO1)in the hippocampus,in order to elucidate its possible mechanisms of action.Methods:1.Experimental animal grouping:The depression mice model was established by chronic unpredictable mild stress(CUMS)for 4 weeks.The probiotics used were a mixture of three strains(Lactobacillus helveticus R0052+Lactobacillus plantarum R1012+Bifidobacterium longum R0175)as freeze-dried stocks.Fluoxetine,a selective serotonin reuptake inhibitor(SSRI),was used as a positive control antidepressant.Male C57BL/6J mice aged 6-8 weeks were randomly divided into 6 groups(CUMS+NS:subjected to the CUMS procedure and received saline,CUMS+F:subjected to the CUMS procedure and received fluoxetine,CUMS+P:subjected to the CUMS procedure and received probiotics,CON+NS:not subjected to any stress and received saline,CON+F:not subjected to any stress and received fluoxetine,and CON+P:not subjected to any stress and received probiotics),with 8 mice in each group.Mice were given corresponding drug intervention using sterile gavage needles once daily.CUMS+P and CON+P groups were fed the probiotics solution 200μL(2×10~9 CFU)daily;CUMS+F and CON+F groups were administered orally with fluoxetine 18 mg/kg body weight daily;CUMS+NS and CON+NS groups were gavaged with equivalent volumes(200μL)of saline solution per day.All CUMS groups were subjected to stress for 28 days(4weeks)at the same time,and all CON groups were not disturbed except for feeding and cage cleaning.2.Behavioral testing:The weight of mice was measured weekly.A battery of behavioral tests,including sucrose preference test(SPT),open field test(OFT),elevated plus maze(EPM)and forced swimming test(FST),was used to evaluate the anxiety-and depressive-like behaviors.3.Fecal microbiota analysis:Cecum contents were sequenced using 16S rDNA,and beta diversity analysis,alpha diversity analysis and species abundance analysis were performed to evaluate the changes in the structure and composition of intestinal microbial communities in the mice.4.Cytokines measurement:The concentrations of cytokines(TNF-α,IFN-γ,IL-6,IL-10)in the serum and hippocampus were quantified using enzyme-linked immunosorbent assay(ELISA).5.Hippocampal indoleamine 2,3-dioxygenase-1(IDO1)protein expression:the expression level of hippocampal IDO1 protein was detected by Western blot analysis.Results:1.CUMS induced significant anxiety-and depressive-like behaviors in mice after 4 weeks.Body weight gain during CUMS was significantly decreased compared with the control group(P<0.05).As measured by SPT the sucrose preference index was decreased significantly by CUMS(P<0.05).In the OFT,the CUMS+NS mice spent significantly less time in the center area than the CON+NS mice did(P<0.05).In the EPM test,a significant decrease of time spent in the open arms was found in the CUMS+NS mice compared with the CON+NS mice(P<0.05).In addition,the CUMS+NS mice were immobile for more time than the CON+NS mice during the FST(P<0.05).And fluoxetine administration completely reversed these CUMS-induced phenotypes.Comparisons with CUMS+NS data showed that:sucrose preference index significantly increased in CUMS+F group(P<0.05);the time spent in the center area in the OFT was significantly prolonged(P<0.05);the time spent in the open arms in the EPM was significantly increased(P<0.05);and the CUMS+F mice exhibited a lower immobility time during the FST(P<0.05).Probiotics treatment partially reversed the anxiety-like behavior in the OFT and EPM and despair behavior in the FST(P<0.05),however the sucrose preference index showed an upward trend without statistical significance.2.Beta diversity analyses displayed that vastly different bacterial community structures existed among the six groups.The microbiota within the CUMS-exposed samples were significantly different than those from the non-stress groups.Theα-diversity index,Chao1 and Simpson reflected the richness and evenness within bacterial populations.We found higher diversity(Chao1,P<0.05)and species evenness(Simpson,P<0.05)after CUMS.In addition,species abundance was analyzed at phylum,class,order,family and genus level.The microbiota composition within all samples belonged to four phyla:Firmicutes,Bacteroidetes,Verrucomicrobia and Proteobacteria,which accounted for 96.94±2.04%of the total sequence.Kruskal-Wallis analysis showed that CUMS had no significant influence on the relative abundance of Firmicutes,Bacteroidetes,Verrucomicrobia and Proteobacteria.At class level,CUMS+NS samples contained lower relative abundance of the Bacilli,and higher relative abundance of Erysipelotrichia and Betaproteobacteria than those of CON+NS samples(P<0.05).At order level,the relative abundance of Lactobacillales in CUMS+NS sample was significantly reduced(P<0.05),while the relative abundance of Erysipelotrichales and Burkholderiales were increased compared with those in CON+NS sample(P<0.05).At family level,CUMS significantly reduced the relative abundance of Lactobacillaceae(P<0.05),while the relative abundance of Ruminococcaceae,Prevotellaceae,Erysipelotrichaceae,Rikenellaceae and Sutterellaceae significantly increased(P<0.05).At genus level,the relative abundance of Lactobacillus and Barnesiella significantly decreased(P<0.05),while the relative abundance of Alloprevotella,Parasutterella and Parabacteroides significantly increased compared with those of CON+NS sample(P<0.05).The probiotics intervention(CUMS+P)significantly increased the relative abundance of class Bacilli,order Lactobacillales,family Lactobacillaceae and genus Lactobacillus(P<0.05),while the relative abundance of family Prevotellaceae,genus Alloprevotella and Parabacteroides were significantly reduced compared with those of CON+NS group(P<0.05).3.Inflammatory cytokine assays showed that after CUMS,serum TNF-αlevel significantly increased(P<0.05),and the hippocampal IFN-γand TNF-αlevels significantly increased(P<0.05).CUMS with supplementation of fluoxetine or probiotics could reduced serum TNF-αand hippocampal IFN-γ,TNF-αlevels(P<0.05).4.Western-blotting results showed that the hippocampal IDO1 expression of CUMS+NS group was significantly higher than that of CON+NS group(P<0.05).Supplementation with probiotics or fluoxetine significantly reduced the hippocampal IDO1 expression.Conclusion:The intestinal bacterial community structure and composition of CUMS mice,as well as the levels of peripheral blood proinflammatory cytokines,hippocampal proinflammatory cytokines and IDO1 protein changed significantly.Probiotics(Lactobacillus helveticus R0052+Lactobacillus plantarum R1012+Bifidobacterium longum R0175)attenuated CUMS-induced anxiety-and depressive-like behaviors,inhibited the serum TNF-αlevel and IFN-γ,TNF-αlevels in the hippocampus by regulating intestinal microecological imbalance,and directly or mediated by inflammatory factors inhibited the high expression of IDO1 in the hippocampus.It is suggested that,the antidepressant-like activity of probiotics(Lactobacillus helveticus R0052+Lactobacillus plantarum R1012+Bifidobacterium longum R0175)may be correlated with the gut microbiota-inflammation-brain axis.