Analysis Of Clostridium Butyricum Function In Ulcerative Colitis And Epithelial-mesenchymal Transition

PART ?Effects of Clostridium butyricum on epithelial-mesenchymal transition in experimental colitisObjective:To investigate the value of live Clostridium butyricum(C.B)in different doses on protecting dextran sodium sulfate(DSS)-induced colitis of mice via regulating epithelial-mesenchymal transition(EMT)Materials and methods:50 male C57B/6 mice were randomly classified into five groups(10 mice per group)as 1)Normal group 2)DSS-induced colitis group 3)Low-dose live C.B treated group 4)Middle-dose live C.B treated group 5)High-dose live C.B treated group.Except mice of normal group,other mice were drinking 3.5%DSS solution every day for 7 days to establish DSS-induced colitis model.Moreover,during 7 days before colitis model,except mice of normal group and DSS-induced colitis group,all other mice were separately received 0.2ml solution with 107CFU/ml,108CFU/ml and 109CFU/ml live bacteria by gastric intubation to construct low-dose live C.B treated group,middle-dose live C.B treated group and high-dose live C.B treated group.All mice were assessed by the following factors(1)The severity of colitis,including disease activity index(DAI)scores,colon lengths,H&E stained scores of colitis and TNF-a concentration in serum by ELISA.(2)EMT of colon tissues in mice,including the expression of E-cadherin(E-cad)mRNA and Vimentin(Vim)mRNA by qRT-PCR and the expression of E-cad protein and Vim protein by Immuno-histochemistry(IHC)staining.All data were presented as means±standard deviation and analysized by SPSS20.0.The statistical differences were tested by one-way analysis of variance(ANOVA)in different groups and by LSD-test in each group.Values of P<0.05 were used as the criterion for statistical significance.Results:Colitis model of mice was established by drinking 3.5%DSS solution for 7 days.Except for one mouse of high-dose live C.B treated group and one mouse of middle-dose live C.B treated group dead accidently in the procedure of experiment,all other mice were survived until the end of experiment.1.The severity of mice colitis(1)In DAI,high-dose live C.B treated group could suppress DAI of DSS-colitis in the early stage of the experiment(at Dayll),but low-dose live C.B treated group and middle-dose live bacteria group exerted this suppression of-DAI in late stage of the experiment(from Day13),P<0.05.(2)In the morphology and colon length,low-dose live C.B treated group and middle-dose live C.B treated group could significantly ameliorate the edema of colon mucosa and the colon length shrink compared to DSS-colitis model,P<0.05.(3)In the injure scores of tissues,both low-dose live C.B treated group and middle-dose live C.B treated group could significantly depress injure scores of colitis,P<0.05.(4)In serum intlammatory factors,all three live C.B treated groups could inhibit the expression of TNF-a in serum,but no statistical siginificance with P>0.05.2.The assessment of EMT in colon tissues of mice(1)By IHC staining,compared to DSS colitis group,all three C.B treated groups could up-regulate the proterin expression of epithelial phenotype E-cadherin without statistical significance(P>0.05)while middle-dose live bacteria group and high-dose live bacteria group could significantly down-regulate the protein expression of mesenchymal phenotype Vimentin(P<0.05).(2)By qRT-PCR,all three C.B treated groups could up-regulate the expression of E-cadherin mRNA in intestinal epithelium compared with the expression in DSS-colitis model without statistical significance(P>0.05),however,middle-dose live C.B treated group could down-regulate the expression of intestinal mesenchymal Vimentin mRNA(P<0.05)Conclusion:1.Evaluation by DAI,colon length,the colonic morphology,colonic tissue injure scores and TNF-a expression in serum,live Clostridium butyricum could ameliorate the severity of DSS-colitis with 108CFU/ml as the best dose.2.In both mRNA and proterin,live Clostridium butyricum in different doses could inhibit the epithelial-mesenchymal transition of colonic tissues in mice by upregulating the epithelial phenotype E-cadherin and downregulating the mesenchymal phenotype Vimentin at the best dose of 108CFU/ml.PART ?Compositions of Clostridium butyricum affect intestinal epithelial cell mesenchymal transition and its cell viabilityObjective:To explore the effect of supernatant and heat-killed Clostridium butyricum in different doses on intestinal epithelial cell mesenchymal transition and its cell viability.Materials and methods:IEC-6 was cultured as control group and conveyed EMT-like transition by 10ng/ml human reconbimant TGF-?1 as positive group while intervented with four groups of potential components of Clostridium butyricum,including supernatant of low-dose(106CFU/ml)and high-dose(107CFU/ml)bacteria,low-dose(106CFU/ml)and high-dose(107CFU/ml)heat-killed bacteria for 4 days.At Day2 and Day4,all cell groups were assessed by(1)Cell morphology under inverted microscope(especial the cell polarity)(2)Cell vability tested with CCK-8 kit.Moreover,all cell groups were demonstrated the effect of EMT at level of mRNA and protein including(1)The expression of E-cadherin,Vimentin and TGF-?1 mRNA by qRT-PCR(2)The expression of E-cadherin and Vimentin protein by Western blotting.In the end,statistical analysis was undergone by SPSS 20.0 with P value<0.05 as the criterion for statistical significance.Results:At Day2,except the significant promotion in high-dose heat-killed C.B group(P<0.05),the cell viability by CCK-8 kit in other groups exhibited no significant difference(P>0.05).At Day4,the cell viability of TGF-?1-induced EMT-like IEC-6 group was decayed compared to control group but promoted by the high-dose bacteria supernatant and dead bacteria with statistical increase by high-dose supernatant of bacteria(P<0.05).In the intestinal epithelial cell mesenchymal transition,(1)Observed by inverted microscope,cell polarity and cell-cell tight-adherin of IEC-6 cultured with TGF-?1 for two days started to be destroyed,however,this could be inhibited by high-dose C.B supernatant and dead bacteria.At Day4,all groups of C.B components could improve cell polarity and cell-cell tight-adherin in TGF-?1 induced EMT-like IEC-6.(2)Tested by qRT-PCR,all bacteria components groups could significantly down-regulate the Vimentin mRNA expression of TGF-?1 induced EMT-like IEC-6 cells(P<0.05)but down-regulate the TGF-?1 mRNA expression without stastical significance(P>0.05),moreover,high-dose C.B supernatant could best up-regulate the E-cadherin mRNA expression(P>0.05).(3)Tested by Western blotting,high-dose C.B supernatant and heat-killed C.B could up-regulate the expression of E-cadherin protein while down-regulate the expression of Vimentin protein in TGF-?1 induced EMT-ike IEC-6.Conclusion:Both C..B supernatant and dead bacteria could inhibit TGF-?1-induced EMT in IEC-6 and increase the cell viability of EMT-like cells with the best effect by 107CFU/ml C.B supernatant.This effect was possible related to abundant short chain fatty acids(SCFAs)in the C.B supernatant.