Effects And Mechanism Of Inulin On Neuroinflammation In Schizophrenia Mice By Regulating Tryptophan Metabolism Via Gut Microbiota

Part one Inulin regulates tryptophan metabolism via gut microbiota on cognitive dysfunction and chronic neuroinflammation in schizophrenia miceRESEARCH PURPOSES1.First,this study aims to explore the effect of Inulin(INU)on amelioration of Schizophrenia(SCZ)-like behavioral and cognitive dysfunction in mice,assess the influence of INU on inflammatory indicators,neurotransmitters,microorganisms,short chain fatty acids(SCFAs)and tryptophan(Trp)metabolism,and clarify the effects of INU on the pathological morphology of the brain and intestine.2.The second purpose is to determine the complex relationships among microorganisms,inflammatory factors,neurotransmitters,SCFAs and Trp metabolites,and to explore how different microorganisms and their metabolites reduce systemic inflammation and inhibit excessive activation of microglia via the microbiota-gut-brain(MGB)axis,thereby reducing neuroinflammation and regulating brain behavioral memory in SCZ mice after INU intervention.RESEARCH METHODSC57BL/6J mice(60,♂)were randomly divided into 4 groups:(a)SCZ group:MK-801(0.6mg/kg body weight)was given intraperitoneal injection for 14 consecutive days and fed a normal diet;(b)INU+SCZ group:After 14 days of intraperitoneal injection of MK-801,the mice were fed drinking water containing INU(2g/kg body weight)for 6 weeks;(c)RIP+SCZ group:14 days after MK-801 administration,mice were fed drinking water containing risperidone(RIP)(0.1mg/kg body weight)for 6 weeks as a positive control group;(d)CON group:An equal volume of normal saline was injected intraperitoneally for 14 days,drinking water normally.After 6 weeks,fresh feces of mice in each group were collected.16S rRNA sequencing was applied to determine differences in fecal flora composition;High-performance liquid chromatography mass spectrometry(HPLC-MS)and gas chromatography mass spectrometry(GC-MS)were performed to test Try metabolites in feces and brain tissue or SCFA in feces.ELISA was applied to determine interleukin(IL)-6,IL-1β,IL-10,tumor necrosis factor-α(TNF-α)in plasma and brain tissue,as well as dopamine(DA),5-hydroxytryptamine(5-HT)and brain-derived neurotrophic factor(BDNF).Immunoturbidimetric method was adopted to detect changes in plasma C-reactive protein(CRP).Limulus reagent was applied to determine changes in plasma lipopolysaccharide(LPS).Flow cytometry was performed to detect the proportion of brain microglia activation.Behavior tests including Open field test(OFT)and Morris water maze(MWM)experiments were separately performed.The immunohistochemistry and Nissl staining of different parts of the brain tissue(hippocampus CA1 area,CA3 area,DG area,and prefrontal cortex)were detected.At the same time,HE staining was applied to show the pathological morphology of the small intestine,and immunohistochemistry was performed to detect the semi-quantitative morphology of intestinal tight junction proteins(Zonula occludens-1,ZO-1 and Occludin).Finally,the correlation analyses were used to evaluate the correlations among different microorganisms and inflammatory factors,neurotransmitters,SCFAs and tryptophan metabolites.These methods were used to prove that INU can exert an influence on the activation of microglia by regulating tryptophan metabolism via gut microbiota through the MGB axis,thus affecting the brain behavior and memory function as well as chronic low-grade neuroinflammation in SCZ mice.RESULTS1.INU could significantly improve the abnormal behavior of SCZ mice(autonomous motor function decline,anxiety disorder,depressive behavior and impairment of learning and memory),and partially reverse the spatial cognitive dysfunction.Compared to the SCZ group,the OFT experiment results indicated that the total distance of autonomous movement,peripheral distance and the number of rare in the INU+SCZ group were distinctly increased.The MWM results also revealed that the mice located the platform much faster in the INU+SCZ group than those in the SCZ group(all P<0.05),meanwhile there was an increase in the number of platform crossings trend in the INU+SCZ mice compared to the SCZ mice but without statistically significant.2.INU had a protective effect on the brain neurons of SCZ mice with significantly increasing the level of BDNF and reducing the expression of 5-HT in the brain(all P<0.05),and the level of DA only showed a downward trend.In Nissl staining,it was observed that the abnormal hyperchromatism of nissl bodies in the various regions of the hippocampus(especially CA3 and DG)in the INU intervention group was sharply reversed compared to the SCZ group.3.INU could effectively reduce systemic inflammation and chronic low-grade neuroinflammation.After INU intervention,plasma LPS,CRP,plasma and brain tissue IL-6,TNF-α,IL-1β,IL-10 were all lower than those in SCZ group(all P<0.05).Brain immunohistochemistry showed that INU could significantly reduce the expression of microglia in the cerebral cortex and hippocampus CA1 and CA3 of SCZ mice(P<0.05).Flow cytometry showed that INU significantly reduced CD11b+ CD45+ and CD11b+ CD45high+ microglia proportions of brain tissue(P<0.05).4.INU increased the expression of ZO-1 and Occludin and improved intestinal permeability(P<0.05).5.16S rRNA high-throughput fecal sequencing revealed that INU intervention had a maj or effect of increasing Bifidobacterium and Lactobacillus while decreasing Akkermansia.Correlation analyses of differential microorganisms with inflammatory cytokines and neurotransmitters showed that Lactobacillus and Bifidobacterium were negatively correlated with brain tissue 5-HT;Akkermansia was found to be positively associated with the above inflammatory cytokines,while negatively associated with BDNF(all P<0.05,absolute value of r>0.46).6.GC-MS showed that INU intervention could reverse the increase of propionic acid in SCFAs of fecal in the SCZ group(P<0.05).7.HPLC-MS displayed that after INU intervention,the levels of cinnabarinic acid(CA)and indole-3-lactic acid(ILA)in tryptophan metabolites in the fecal and brain tissue were both significant increased,and the content of quinolinic acid(QA)was significantly decreased(all P<0.05).8.After INU intervention,the correlation analyses among SCFAs,tryptophan metabolites and differential gut microbiota indicated that SCFAs except propionic acid were positively associated with ILA.SCFAs showed a negative correlation trend with Akkermansia and Negativibacillus.Among them,valeric acid,isobutyric acid and isovaleric acid were distinctly negatively correlated with Akkermansia.Among the tryptophan metabolites,CA was positively associated with Bifidobacterium,Butyricicoccus,Rikenellaceae-RC9-gut-group and unclassified-f-ⅩⅢ,but negatively associated with Akkermansia,Erysipelatoclostridium,Negativibacillus,Anaeroplasma,Candidatus-Arthromitus,Roseburia and unclassified-fRikenellaceae.ILA was positively associated with Erysipelatoclostridium,Rikenellaceae-RC9gut-group and Turicibacter(all P<0.05,absolute value of r>0.54).9.After INU intervention,SCFAs,tryptophan metabolites and inflammatory cytokines were closely related.The above brain inflammatory cytokines were positively associated with propionic acid while negatively associated with caproic acid.CA in tryptophan metabolites was negatively correlated with the above-mentioned inflammatory cytokines,and ILA was significantly negatively correlated with TNF-α(both P<0.05,absolute value of r>0.58).CONCLUSIONSDietary INU inhibits chronic low-grade neuroinflammation via regulating tryptophan metabolism by gut microbiota through the MGB axis,thereby partial improving the behavior and cognitive dysfunction in SCZ mice.Part two The effect and mechanism of tryptophan metabolite cinnabarinic acid on inhibition of neuroinflammation in BV-2 cellsRESEARCH PURPOSES1.First,this part of the study aims to explores the inhibitory effect of Trp metabolite cinnabarinic(CA)on the excessive activation of BV-2 cells induced by LPS,and to evaluate its influence on inflammation indicators and the polarization of microglia M1/M2.2.The second purpose is to clarify the anti-inflammatory molecular mechanism of CA on the excessive activation of BV-2 cells induced by LPS.After the corresponding interventions of pathway proteins or receptors are given,inflammatory cytokines and the expression of pathway proteins or receptors should be evaluated.we stimulated microglial BV-2 cells with low concentration of LPS to establish neuroinflammation model in vitro,so as to clarify the possible mechanism of inulin regulating tryptophan metabolism through gut microbiota and inhibiting neuroinflammation in schizophrenia mice.RESEARCH METHODS1.We stimulated microglial BV-2 cells with low concentration of LPS to establish neuroinflammation model in vitro.The BV-2 cells were randomly divided into a CON group,a LPS group,a LPS+CA group and CA group to study the anti-inflammatory effect of CA on microglia induced by LPS.The CCK-8 kit was performed to measure the viability of BV-2 cells with different concentrations(10μM,30μM,100μM and 300μM)of CA,and to determine the optimum of CA that did not affect cell viability.A nitric oxide(NO)kit was applied to test the concentration of NO in the cell supernatant for determining the optimum time for CA to stimulate the cells.ELISA was applied to determine the changes of inflammatory cytokines(IL1β,IL-18 and TNF-α).Immunofluorescence labeling of inducible nitric oxide synthase(iNOS)and arginase 1(Argl)were applied to observe the polarization of M1/M2 of BV-2 cells.2.Based on BV-2 cells stimulated by LPS and given the optimum concentration of CA intervention,the metabolic glutamate receptor 4(mGluR4)antagonist a-Methyl-4-phosphonophenylglycine(MPPG),the adenylate cyclase(AC)agonist Forskolin(FSK)and protein kinase A(PKA)inhibitor N-[2-(p-bromocinnamyl amino)ethyl]-5-isoquinoline sulfonamide,dihydrochloride(H89)were given respectively.ELISA experiments were performed to measure changes in the above inflammatory cytokines.The cyclic adenosine monophosphate(cAMP)kit was applied to test intracellular cAMP levels.Western blot was adopted to show the expression of proteins such as mGluR4,PKA,nuclear factor kappa-B p65(NF-κB p65)and nucleotide oligomerization domain like receptor proteins family pyrin domain proteins 3(NLRP3)in BV-2 cells at the protein levels.RESULTS1.The results of CCK8 indicated that after 300μM CA administration in BV-2 cells stimulated by LPS for 24h as well as 100μM and 300μM CA intervention for 48h,cells viabilities were significantly reduced(all P<0.05).BV-2 cells stimulated by LPS were given 10μM,30μM,100μM CA intervention for 24h,which could distinctly decrease the lever of NO released,and lasted to 48h(all P<0.05),thus 24h was recognized as the optimum time for the following experiment.2.ELISA showed that compared with 10μM and 100μM CA administration,the concentrations of IL-18,IL-1β and TNF-α were distinctly decreased in 30μM CA administration group than those in LPS group(all P<0.05).Combined with NO release and CCK8 detection results,the optimum CA concentration was decided to be 30μM.In addtion,western blot of NF-κB p65 in 30μM CA administration group also showed similar trend(P<0.05).3.Immunofluorescence qualitative analysis indicated that 30μM CA administration could inhibit the increase of Iba1 expression in BV-2 cells induced by LPS,and activate mGluR4 on BV-2 cells.4.Immunofluorescence double labeling showed that 30μM CA administration could sharply reverse the high expression of iNOS in the LPS group(P<0.05),but without obvious distinction in the Argl between the LPS+CA group and LPS groups.5.After MPPG,FSK and H89 were given respectively to 30μM CA intervention group,compared with the LPS+CA group,the releases of IL-18 and IL-1β from BV-2 cells were significantly increased(all P<0.05).However,it was found that the mGluR4 antagonist MPPG could not completely inhibit the anti-inflammatory effect of CA in the determination of TNF-αcompared with the expression of LPS+CA group,there was only an upward trend.6.The trend of cAMP in BV-2 cells was similar to that of inflammatory cyotokins,that was,after MPPG,FSK and H89 treatments,the intracellular cAMP was significantly increased in the CA(30μM)intervention group(all P<0.05).7.Western blot detection was performed on mGluR4,PKA,NF-κB p65,NLRP3,respectively,and the results indicated that 30μM CA could activate mGluR4 and drastically reduce the high expression of PKA,NF-κB p65 and NLRP3 induced by LPS.After MPPG,FSK and H89 intervention,PKA and NLRP3 were dramatically higher than those in LPS+CA2 group(all P<0.05),while NF-κB P65 only increased without statistical significance.CONCLUSIONSIn the neuroinflammation model of BV-2 cells induced by LPS,the Trp metabolite CA activates mGluR4 to inhibit the AC-cAMP-PKA-NF-κB/NLRP3 pathway,thereby reversing the excessive activation of microglia and making it polarized from M1 to M2 to alleviate neuroinflammation.