Study On The Protective Effect Of Probiotics On Intestinal Epithelial Cell Junction In Colitis

Part Ⅰ.Protective effects of probiotics on intestinal epithelial cellular junctions in mice under colitis.Objective:To investigate the effects of probiotics(Bifidobacterium,Lactobacillus),prebiotics(lactitol)and synbiotics(Bifidobacterium + Lactobacillus + lactitol)on cellular junctions of intestinal epithelial cells in mice with DSS-induced acute colitis.Methods:Male C57BL/6J mice were divided into 5 groups.All but the blank control group were administered DSS solution.The DSS model control group was not treated with gavage,while the other 3 groups were given probiotics(Bifidobacterium,Lactobacillus).Prebiotics(lactitol),and synbiotics(probiotics combined with lactitol)respectively.After gavage for 1 week,colon specimens were collected and examined for:pathological inflammation scoring;transmission electron microscopy;immunohistochemical staining of Occludin,ZO-1,E-Cadherin and β-catenin;Western blot analysis of Occludin,ZO-1,and E-cadherin proteins;qRT-PCR analysis of Occludin,ZO-1,and E-cadherin mRNA.Results:1.The pathological inflammation scores of mice administered probiotics,prebiotics and synbiotics were significantly lower than those of the DSS model group.2.Transmission electron microscopy showed that mice in the probiotics,prebiotics and synbiotics groups show less relaxation of adherens junctions and less cell shedding,significantly prolonged tight junctions in shedding epithelials,and less relaxation of adherens junctions in shedding epithelial cells,in comparison to the DSS model group.3.Western blot showed relatively higher concentrations of Occludin,ZO-1,and E-Cadherin proteins in colic tissues in the probiotics,prebiotics,and synbiotics groups compared to the DSS model group.4.qRT-PCR showed that the expression levels of E-cadherin and ZO-1 mRNA were significantly higher in the probiotics,prebiotics and synbiotics groups than in the DSS model group.Conclusion:Probiotics,prebiotics and synbiotics can:1.Significantly reduce the degree of inflammation and intestinal epithelial destruction in mice with DSS-reduced acute colitis;2.Reduce the destruction of adherens junctions in mice with acute colitis,and promote the prolongation of tight junctions,along with intensification of adherens junctions,in the advent of cell shedding;3.Increase the relative concentration of Occludin,ZO-1 and E-cadherin proteins in colic tissues of mice with acute colitis;4.Up-regulate levels of E-cadherin and ZO-1 mRNA transcription in mice with acute colitis.Part Ⅱ.Protective effects of Saccharomyces boulardii and Bifidobacterium on cellular junctions of cells under proinflammatory factor stimulationObjective:Through co-culture of probiotics and cells,investigate the protective effects of probiotics Saccharomyces boulardii and Bifidobacterium on the cellular junction of Caco-2 cell monolayers under stimulation of proinflammatory factor IL-6.Methods:Caco-2 cell monolayers were divided into 8 groups,respectively co-cultured with S.boulardii,Bifidobacterium,Escherichia coli,no co-culture,and another 4 of the aforementioned co-cultures with the proinflammatory factor IL-6 added.Immunofluorescence staining,Western blot,co-immunoprecipitation and qRT-PCR were used to study the localization,relative content,relative binding affinity and mRNA transcription level of Occludin/ZO-1 and E-cadherin/β-catenin junction complexes.Results:1.Immunofluorescence results showed that in Caco-2 cells co-cultured with Bifidobacterium,IL-6 stimulation did not induce neucleation of β-catenin and dislocation of E-Cadherin/p-catenin and Occludin from cell membranes,as opposed to cells co-cultured with E.coli;2.Western blot showed that with no co-culture and co-culture with E.coli,the level of E-Cadherin and ZO-1 protein decreased after the addition of IL-6 to Caco-2 cells,while the proteins did not decrease after the IL-6 was added to cells co-cultured with S.boulardii and Bifidobacterium;3.The results of immunoprecipitation showed that the ratio of E-Cadherin co-precipitated with β-catenin was not significantly decreased after stimulation with IL-6 in the co-culture group with S.boulardii and Bifidobacterium,while there was a significant decrease in co-culture with E.coli;qRT-PCR results showed that IL-6 stimulation significantly up-regulated the transcription levels of Occludin,E-Cadherin and ZO-1 mRNA in co-cultured cells with S.boulardii and Bifidobacterium,with statistically significant diffenrences.Conclusions:1.Bifidobacterium can reduce the P-catenin nucleation of Caco-2 cells stimulated by IL-6,maintain the co-localization of E-Cadherin/β-catenin in the cell membrane and the membrane localization of Occludin;2.S.boulardii and Bifidobacteria can inhibit the decrease of E-Cadherin and ZO-1 protein levels in IL-6-stimulated cells;3.S.boulardii and Bifidobacterium can inhibit the decrease of E-Cadherin binding with P-catenin after stimulation by IL-6;4.IL-6 stimulation up-regulated the transcription levels of Occludin,E-Cadherin,and ZO-1 mRNA in cells co-cultured with S.boulardii and Bifidobacterium.