| Part 1 Influence of prebiotics on intestinal microflora in normal mice and mice with acute colitisObjective:To investigate the effects of probiotics（Bifidobacterium and Lactobacillus）,prebiotics（Lactitol and lactulose）,and their combination on enteric flora of normal mice and mice of acute colitis.Methods:1.Effects on intestinal microflora of normal mice.C57BL/6J mice were randomly divided into 7 groups.Except for control group,other groups were gavaged with probiotics,lactitol of standard concentration,lactitol of high concentration,probiotics and lactitol,lactulose,probiotics and lactulose for two weeks,respectively.The feces of before gavage,gavage for 1 week,gavage for 2 weeks and intestinal mucosa were collected to analyze intestinal microbiota through 16s rDNA sequencing.2.Effects on intestinal microflora of mice with acute colitis.C57BL/6J mice were divided into 6 groups randomly.Each group was given 2.5%DSS drinking water for 5 days other than the blank control group.Except for model control group,other four groups were intervened with probiotics,probiotics and inulin,lactitol,probiotics and lactitol.Half of the mice were sacrificed after 1 week of gavage and the remaining mice were killed after gavage for 10 days.According to the pathological results,samples were determined for deep analysis.The feces of different periods and intestinal mucosa samples were collected to analyze the differences of intestinal microbiota by 16s rDNA sequencing.Results:1.Compare with other groups,feces and mucosa of lactitol group showed significant increase of Akkermansia.Lactulose could also promote the proliferation of Akkermansia,but with less obvious effect than lactitol.There was no statistically significant difference between the two groups with different concentration of lactitol.2.After gavage for 1 week,the pathological scores of groups with intervention were significantly lower than those in the model control group,and the difference was statistically significant.3.The model control group was higher in the genus of Bacteroides and lower in Lactobacillus,Roseburia compared with blank group.However,the same phenomenon was not found in groups gavaged with probiotics and lactitol.Compared with model control group,mice with intervention were increased with Bifidobacterium,LachnospiraceaeNK4A136group,LachnospiraceaeUCG-006 and decreased with Alistipes in varying degrees.Unlike normal mice,the proliferation of lactitol on Akkermansia was not obvious among mice with acute colitis.4.The mucosal flora was more abundant than the fecal flora,and genus of Mucispirillum was more common in mucusa.Conclusions:1.Prebiotics can regulate intestinal microflora,and the effects of different types are not the same.Lactitol could promote the proliferation of Akkermansia more significantly than lactulose.2.Probiotics and lactitol reduced the degree of inflammation in acute colitis mice obviously.3.Mice with acute colitis showed reduced beneficial genera and increased harmful genera.Supplementation of probiotics and prebiotics displayed advantage of increasing the proportion of helpful bacteria and regulating the balance of intestinal microbiota.Part 2 Mechanism of Bifidobacterium and Saccharomyces boulardii on the course of colitis related carcinogenesisObjective:Through co-culture probiotics and cells,to investigate the mechanism of probiotics in the process of colitis related carcinogenesis at the cell level.Methods:1.Caco-2 cells were co-cultured with supernatants and precipitates of Bifidobacterium respectively,and stimulated by IL-6 and TNF-α,then the relative luciferase activity was detected by double luciferase reporter assay.The experiment was repeated three times,and co-culture mode was selected according to the results.2.Co-culture Caco-2 cells,CCC-HIE-2 cells with Bifidobacterium and Saccharomyces boulardii respectively.Set 6 groups,namely blank control group,probiotics group,IL-6 group,probiotics plus IL-6 group,TNF-a group,probiotics plus TNF-a group,measured the relative luciferase activity.Different methods such as qRT-PCR,Western Blot,ELISA and EMSA were operated to detect the level of mRNA and protein of β-catenin,the expression of β-catenin in nucleoprotein,the level of inflammatory factors,the transcriptional activity of NF-κB and TCF-4.Results:1.Co-cultured Caco-2 cells with Bifidobacterium of different volume,whether added IL-6 or TNF-a or not,relative luciferase activity may be increase,decrease or change without statistical significance compared with other groups.On the contrary,when co-cultured with precipitates,the relative luciferase activity decreased significantly compared with corresponding group.Results of three times showed a high degree of consistency.Therefore,we chose co-culture probiotics precipitation with cells in our following experiments.2.Four co-culture combinations showed consistent experimental results.Compared the six groups,there was no significant difference in the expression ofβ-catenin mRNA and protein.Compared probiotics group with blank control group,probiotics plus IL6 group with IL-6 group,probiotics plus TNF-α group with TNF-αgroup,the level of IL-6 and TNF-αwas down-regulated,and difference was statistically significant.At the same time,the binding band of NF-κ B and DNA,TCF-4 and DNA decreased obviously,the expression level of β-catenin in nucleus was distinctly declined.Conclusions:1.Probiotics precipitation co-cultured with cells was a better method compared with supernatant.2.Co-culture Caco-2 cells,CCC-HIE-2 cells with Bifidobacterium and Saccharomyces boulardii could inhibit the activity of Wnt signaling pathway,which mechanism included down-regulate the level of inflammatory factors,reduce the transcriptional activity of NF-κ B and TCF-4,and inhibit the process of entering nucleus from cytoplasm for β-catenin. |
