The Mechanism Of Probiotics To Protect The Barrier Function Of Intestinal Mucosa

Part 1Objective:To investigate the protective effect of probiotic VSL#3 and sodium butyrate(SB)on intestinal mucosal barrier function in mice with acute enteritis induced by Dextran Sulfate Sodium(DSS),and the regulatory effect of these two substances on cell junction proteins and TFF-3.Method:Male C57BL/6J mice were divided into 5 groups:the control group,the DSS group,the DSS+VSL#3 group,the DSS+SB group,and the VSL#3 group.In addition to the blank control group and the VSL#3 group,the rest of the groups were treated with DSS to establish the acute enteritis model and were given water,VSL#3 and SB by gavage,respectively.The VSL#3 group was given VSL#3 by gavage.After gavage for 1 week,the disease degree of mice were assessed by disease activity index(DAI)and colon length.The intestinal mucosal barrier function of mice was evaluated by intestinal mucosal permeability test.Colon specimens were collected and colon inflammation was assessed by hematoxylin-eosin staining and inflammation scoring.The transcription levels and relative protein contents of Occludin,ZO-1,E-cadherin,β-catenin and TFF-3 were assessed by qRT-PCR and Western blot.Immunohistochemical staining was used to observe the contents and distribution of Occludin,ZO-1,E-cadherin and β-catenin.The feces of mice were collected and the short chain fatty acid content was detected by gas chromatography.Results:after 1 week of treatment,colon length of mice from the DSS group or the DSS+SB group was significantly shorter than that of the control group(the former:P=0.000,the latter:P=0.019),and the colon length of DSS mice with VSL#3 or SB gavage was significantly longer than that of DSS mice without intervention(the former:P=0.002,the latter:P=0.011).After VSL#3 or SB gavage,the DAI scores of DSS mice were significantly lower than that of DSS mice without intervention(The former:P=0.046,The latter:P=0,032),and their pathological inflammation scores were also significantly lower than that of the DSS mice without intervention(the former:P=0.000,the latter:P=0.000).The intestinal mucosal permeability of DSS mice with or without VSL#3 or SB gavage was significantly higher than that of the control group(non-intervention:P=0.000,VSL#3 gavage:P=0.001,SB gavage:P=0.001),but the intestinal mucosal permeability of DSS mice treated by VSL#3 or SB intervention was significantly lower than that of DSS mice without intervention(the former:P=0.000,the latter:P=0.000).There was no significant difference in the above indexes between the VSL#3 group and the control group.The mRNA levels and relative protein contents of Occludin,ZO-1 and E-cadherin in the DSS group were significantly lower than those in the control group.After VSL#3 or SB gavege,the transcription level and the relative protein content of Occludin and/or ZO-1 in DSS mice increased significantly,and the relative protein content of E-cadherin also increased evidently.The level of β-catenin did not show significant difference among groups.The mRNA levels of TFF-3 in DSS mice with or without VSL#3 or SB gavage were significantly increased.The result of immunohistochemical showed that Occludin,ZO-1,E-cadherin and β-catenin proteins of the DSS group dissociated from the cell membrane and shifted into the cytoplasm,while VSL#3 and SB gavage could promote the above proteins to relocate to the cell membrane.The results of gas chromatography showed that the relative content of butyric acid in feces of mice was significantly higher in the VSL#3 group compared to that of the control group(P=0.017),the DSS group(P=0.001)and the DSS+VSL#3 group(P=0.003).Conclusion:VSL#3 and Sodium butyrate gavage can alleviate the acute colitis caused by DSS in mice and reduced the intestinal mucosal permeability.They can enhance the expression of Occludin,ZO-1 and E-cadherin,and promote the redistribution of Occludin,ZO-1,E-cadherin and β-catenin from cytosol to cell membrane in DSS mice.VSL#3 increased the relative amount of fecal butyric acid in healthy mice.Part 2Objective:To investigate the protective effect and the possible mechanism of sodium butyrate(SB)on the integrity of Caco-2 monolayer cells under stimulation of interlukin-6(IL-6).Methods:Caco-2 cells were divided into four groups:the control group,the IL-6 group,the IL-6+SB group(pretreated with SB before IL-6 stimulation)and the SB group.The barrier function of Caco-2 monolayer was assessed by measuring the transepithelial electrical resistance(TEER)and cell monolayer permeability.The transcription levels and relative protein contents of Occludin,ZO-1,E-cadherin,(3-catenin and TFF-3 were detected by qRT-PCR and Western blot.The distribution of cell junction proteins in cells was detected by immunofluorescence staining.The relative binding affinity of cell junction proteins was detected by co-immunoprecipitation(co-IP).The scratch test was used to evaluate the effect of SB and TFF-3 on wound healing of monolayer cells.Results:IL-6 stimulation significantly reduced the TEER of Caco-2 monolayer cells(P=0.000)and increased the permeability of monolayer cells(P=0.001)compared with the control group,while SB pre-treated increased the TEER(P=0.000)and decreased the permeability of the monolayer cells(P=0.035)compared with the non-intervention IL-6 group.IL-6 and SB co-treatment could increase the transcriptional level of E-cadherin,while there was no significant difference in mRNA levels of other proteins among groups.The relative protein contents of Occludin,ZO-1 and E-cadherin of Caco-2 decreased significantly after IL-6 stimulation,and the decrease of Occludin and ZO-1 could be retarded by SB intervention.Little difference was observed on the relative protein contents of β-catenin and TFF-3 among groups.Immunofluorescence staining showed that IL-6 stimulated the migration of junction proteins from the cell membrane to the cytoplasm,and SB could promote the redistribution of these proteins to the cell membrane.The result of co-IP showed that the binding affinity of Occludin and ZO-1,as well as that of E-cadherin and β-catenin decreased after IL-6 stimulation,while SB treatment could reverse this trend.The scratch test showed that SB could promote the wound healing of monolayer cell in the presence of TNF-a through enhancing the migration of Caco-2 cells in a dispersed pattern,while TFF-3 could promote wound healing by inducing cell migration in a continuous pattern.Conclusion:Sodium butyrate can protect the barrier function of monolayer Caco-2 cells under IL-6 stimulation.It can increase the expression of Occludin and ZO-1,promote the redistribution of Occludin,ZO-1,E-cadherin and p-catenin from cytoplasm to the cell membrane,and increased the binding affinity of junction proteins.SB and TFF-3 can both promote cratch healing,but their effects follow different patterns.