| Saccharomyces boulardii is a kind of probiotic yeast isolated from the fruit of Indonesia litchi by Henri Boulard,a French scientist in 1920.In Europe,Africa and other areas,Saccharomyces boulardii(Sb)has been used widely in the prevention and treatment of infantile diarrhea,traveler diarrhea,and the recovery and balance of the intestinal environment after intestinal surgery.Even though it is one of the most valuable boulardii probiotics,the present research of Sb is mainly focused on the production and clinical use,and few studies have been done and reported on the effect of its anti-inflammatory and immunoregulatory molecular mechanism as well as the genetic transformation to improve the probiotic effect.Therefore,we selected the VRP1 gene as the research object to study the anti-inflammatory effect of yeast in order to improve its effectiveness.We knocked out the VRP1 gene in the test and constructed the mutant strain because of high content of ?-glucan to compare the phenotype of Sb-thp1 and Sb-vrp1,including the shape and size,the cell wall structure,the growth speed.Then the safety of two strains of bacteria was evaluated.In final,we build DSS enteritis model test to study the mutants in the prevention of enteritis.Better probiotic strains were selected so as to discuss the variation of probiotic mechanism.1.Construction of Sb VRP1 strains with gene absentYeast beta glucan,as a good natural immune enhancer for the non-specific and specific immune function of airframe,accounts for more than 50% of female bacteria cell wall.Therefore,Sb strains with high content of beta glucan were constructed and selected to enrich the natural resources of beta glucan and broaden the application of Sb.Our lab used Sb Cre-lox P knockout system to completely delete the VRP1 gene from the yeast genome.Then,we used PCR identification to knock out strains and gained variants with missing VRP1 gene.2.Phenotype of Sb and its variants with missing geneWe did a comparable study of phenotypes of wild Sb strains and its variants in the shape size,cell wall composition,growth speed,cell wall sensitivity,tolerance to outside artificial gastric acid and bile salt,as well as retention in mice.Under scanning electron microscope,Sb-thp1 became round,while Sb-vrp1 became long and thin,compared with Sb.Compared with Sb and Sb-thp1,the thickness of Sb-vrp1 cell wall increased by 13.4% and 10.7% respectively,the single-layer thickness of mannan increased by 25.1% and 19.5% respectively,and single-layer thickness of Glucan increased by 10.9% and 4.2% respectively,under transmission electron microscope.Compared with Sb,Sb-vrp1 grew at the same speed,while Sb-thp1 grew at a lower speed.There was no difference between Sb,and Sb-thp1 and Sb-vrp1 on the sensitivity to Congo red.In experiments testing resistance to hydrochloric acid of different pH,less significant differences were discovered on survival rate.In experiments testing resistance to bile salt of different concentrations,the survival rate of Sb-vrp1 was significantly higher than that of Sb and that of Sb-thp1.In experiments testing the retention in mice,the retention time of Sb-vrp1 was 8 hours longer than that of Sb and Sb-thp1 was 12 hours longer than Sb.To sum up,the phenotype of Sb was influenced to some extent if VRP1 and THP1 genes were knocked out.3.The safety assessment of Sb-vrp1 and Sb-thp1 gene-deleted stains.We assessed the safety of Sb-vrp1 and Sb-thp1 gene-deleted stains by acute toxicity test and chronic repeated test.And it was found in one-off large dose(2×109 strains)acute toxicity experiment that the stains of Sb-vrp1 and Sb-thp1 gene-deleted had no obvious differences with normal control in clinical manifestation,body weight change,blood parameters etc.,and the body has not shown the disease of toxic effects.Sb-vrp1 and Sb-thp1(2×108 strains),medium(1×108 bacteria),and low(2×107 bacteria)were used to carry on the mice every day for a period of twenty-eight days.Compared with the control group,there were no significant differences in the clinical manifestations,body weight changes,blood routine and blood biochemistry in each dose group,and all of them were in the normal range.In addition,the dose of the same strain had no significant effect on the normal physiological and blood parameters,and the same dose of different strains showed no significant difference.Therefore,it can be concluded that Sb-vrp1 and Sb-thp1 are safe for mice.4.Construction of DSS colitis modelDSS colitis model is similar to human inflammatory bowel disease and it is widely used because it is reproducible and easy to operate.Animal model of DSS colitis was constructed by selecting C57BL/J six-week female mice as the experimental animals in order to do further research of the DSS using dose and test indicators and to explore the best test condition,which aimed to study Sb and its functions to prevent and treat colitis.The results showed that drinking DSS of 3% for 8 days could construct the DSS animal model of acute colitis;the disease activity index and pathological histology scores could be viewed as the evaluation standard of DSS colitis.Disease activity index is the average number of scores of weight loss and intestinal bleeding.When the weight rises or does not change,the score of weight loss is zero.When weight loses by 1 ? 5%,the score is 1;when weight loses by 5 ? 10%,the score is 2;when weight loses by 10 ? 15%,the score is 3;when weight loses more than 15%,the score is 4.The intestinal bleeding score is 0 under normal stool consistency with negative hemoccult,1 under soft stools with positive hemoccult,2 under very soft stools with traces of blood,3 under watery excrement stools with visible rectal bleeding and 4 under gross bleeding.Histopathological scoring is based on invasion of lymphatic cells and damage of epithelium mucos.The score is 0 with normal situation,1 with slight increase in cellularity,2 with increased cellularity including neutrophils,mild edema,3 with focal erosions,ulcerations of the mucosa,4 with large and multifocal mucosal ulcerations,and 5 with loss of mucosal architecture.5.Preventive effects of Sb and its variants on DSS colitisIn our DSS colitis model,four-weeks C57BL/6J female mice drank 108 Sb by gavage for 22 days in a row.On the 14 th day,DSS of 3% was used for 8 days to induce colitis.Then the effects of Sb and the variants were observed on preventing colitis and the influences on mice's inflammatory factors and barriers of relative gene expression and immune system.It was found that Sb-vrp1 and Sb-thp1 could reduce DSS colitis disease activity index and colon pathological tissue injury and alleviate the colon lesions to make the spleen index and MPO enzyme activity close to the control group.Through RT-PCR detection,it was found that Sb-vrp1 could reduce proinflammatory factors such as IL-6,IL-17,iNOS,TNF-?,MMP-2 and MMP-9,and could raise intestinal barrier such as MUC-2,ZO-1 and Occludin,which could achieve the prevention and treatment effects by reducing inflammation and strengthening the barrier function of bowel wall.In addition,Sb and its variants could adjust antibody levels.could make normal mice SIg A concentration rise by 175% and make SIgA concentration in mice with enteritis rise by 61.7%.Meanwhile,Sb-vrp1 had an influence on the proportion of lymphocytes of mesenteric lymph nodes.In DSS colitis,Sb-vrp1 could effectively cut the proportion of B lymphocytes and CD4+CD25+T lymphocyte cells in MLN,and raise the proportion of T lymphocytes,CD8+T lymphocytes subgroup and CD4+T lymphocyte subsets,which made a contribution in maintaining the immune balance and stability.These results demonstrated that Sb and the variants had a certain influence on celluler and humoral immunity of mice,and could remit DSS enteritis by SIgA and changing T lymphocytes and its subgroups in mesenteric lymph nodes. |
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