| Objectives The effects of manganese and high-fat diet on gut microbiota and its role in nerve injury are not clear.The study is aimed to investigate the effects on neurobehavior and neuroinflammation and changes of gut microbiota,SCFAs and Tregs cells in high-fat diet mice exposed to manganese.To clarify the improved effects of the nerve injury of HFD mice exposed to manganese by regulating the gut microbiota Bacteroides thetaiotaomicron(BTeta)and Clostridium butyricum(CBut).Methods 1 80 Male adult SPF C57BL/6 mice were randomly divided into control group,high-fat diet group(HFD group),manganese exposure group(Mn group)and high-fat diet + manganese exposure group(HFD+Mn group).Mice in control group and Mn group were fed normal diet,while the HFD group and HFD+Mn group were fed high-fat diet.The Mn group and HFD+Mn group were given 10mg/kg/d Mn Cl2 for 12 weeks by intraperitoneal injection.The motor coordination,exploratory behavior and anxiety state of mice were tested by rotated test,open-field test and elevated plus maze test.The injury was observed by HE staining,and the protein expressions of IL-6,TNF-α,IL-10 and Foxp3 were determined by Western blot in striatum.Real-time PCR was used to detect the specific Treg genes PD-1,Ctla4,Helios and Gitr in striatum,as well as the tight junction proteins ZO-1,claudin-5,and occludin m RNA levels.2 Diversity and relative abundance of gut microbiota were measured by 16 S r DNA sequencing technology.SCFAs contents(acetic acid,propionic acid,butyric acid,etc.)were detected by GC-MS.The protein expressions of IL-6,TNF-α,IL-10 and Foxp3 in colon were determined by Western blot.Real-time PCR was used to analyze the m RNA levels of Gpr43 and Gpr109 a.Protein levels of IL-10 and TGF-β1 in mouse serum were detected by ELISA.The changes of Treg/CD4+T percentage in blood were measured by flow cytometry.3 Effects of BTeta and CBut on nerve function and neuroinflammation in mice exposed to Mn and high-fat diet were investigated.Male SPF C57BL/6 mice were randomly divided into two groups,20 were given basic food as control group.The remaining 60 were given HFD and 10mg/kg/ day Mn Cl2 for 12 weeks.Then,60 HFD+Mn mice were randomly divided into HFD+Mn exposure group(HM group),BTeta-treated group(HM+BTeta group)and CBut-treated group(HM+CBut group).They were treated 1×108/d cfu BTeta and CBut by gavage for 4 weeks.Subsequently,protein expressions of IL-6,TNF-α,IL-10 and Foxp3 in colon and striatum,m RNA levels of PD-1,Ctla4,Helios and Gitr in striatum,and gut microbiota relative abundance and SCFAs were detected,respectively.Results 1 Compared to HFD group,lantencys in rotate test,time in central,crossing,total distance and rearing in open field test,and percent of time in open arms,number of enters to open arms of HFD+Mn mice lower(P<0.05).Pathological damage of striatum in HFD+Mn mice were worst.Compared with other groups,the protein expression levels of IL-6 and TNF-α were increased significantly in striatum of HFD+Mn mice(P<0.05),whereas IL-10 and Foxp3 were reduced markedly(P<0.05).Moreover,the m RNA expression levels of PD-1,Ctla4 and Helios in striatum of HFD+Mn mice were lower than control and HFD group(P<0.05).Meanwhile,The same consequence happened in claudin-5 and occludin m RNA of the striatum in HFD+Mn mice(P<0.05)。All above differences were statistically significant.2 Diversity of gut microbiota in HFD+Mn mice were decreased,and relative abundance ratio of Firmicutes/Bacteroidetes were increased than other groups.The relative abundance of Oscillospira,Bacteroides,Prevotella,Akkermansia and Roseburia were decreased obviously(P<0.05),whereas the relative abundance of Lactobacillus were increased(P<0.05).Meanwhile,the content of acetic acid,propionic acid and butyric acid of HFD+Mn mice were lower than those of HFD group(P<0.05).The protein expression levels of IL-6 and TNF-α were increased in the colon of HFD+Mn mice(P<0.05),and IL-10 and Foxp3 were decreased prominently(P<0.05).Moreover,the m RNA expression levels of SCFAs receptor genes Gpr43 and Gpr109 a in the colon of HFD+Mn mice were declined(P<0.05).In addition,the percentage of Treg /CD4+T in blood of HFD+Mn mice descended,and the protein expression levels of IL-10 and TGF-β1 in serum were also lower than those in the HFD group notably(P<0.05).All above differences were statistically significant.3 Compared to HM group,the gut microbiota taxas in HM+BTeta group and HM+CBut group were increased.The relative abundance ratio of Firmicutes/Bacteroidetes were decreased,and the relative abundance of Prevotella,Oscillospira,Bacteroides and Roseburia were ascended(P<0.05),while Lactobacillus were descended markedly(P<0.05).The contents of acetic acid and propionic acid of HM+BTeta mice butyric acid of HM+CBut mice were rised obviously(P<0.05).Compared with the HM group,the protein expression levels of IL-6,TNF-α were reduced and IL-10,Foxp3 were augmented by BTeta and CBut intervented in colon(P<0.05).In addition,the m RNA expressions of Gpr43 in HM+BTeta group,and Gpr109 a of HM+CBut group were significantly higher than those of HM group in colon(P<0.05).Compared to HM group,neurobehavior factors of HM+BTeta or HM+CBut group were changed(P<0.05).The protein expression levels of IL-6,TNF-α were declined,and IL-10 and Foxp3 were rised(P<0.05)in strituam of HM+BTeta and HM+CBut group.Meanwhile,the m RNA expression levels of PD-1,Ctla4,Helios and Gitr were elevated in strituam of HM+BTeta and HM+CBut(P<0.05).All above differences were statistically significant.Conclusions 1 Nerve injury(neurological dysfunction,neuroinflammation)and Tregs function of HFD mice were exacerbated by manganese-exposed.2 The abnormal gut microbiota and SCFAs of HFD+Mn mice were affected.3 BTeta and CBut regulate Tregs level andfunction by gut microbiota,SCFAs and receptor gene Gpr43 and Gpr109 a,then relieve neurological dysfunction and neuro-inflammation,providing a basis of preventing and treating nerve injury by microbial in clinical.Figure 31;Table 20;Reference 301... |
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