| Objective:Cystic echinococcosis(CE)is a serious worldwide zoonosis caused by Echinococcus granulosus(E.g).Human is an intermediate host infected by ingesting the eggs of Echinococcus from carnivorous faeces.With the prolongation of course,the disease seriously affects on the health of population in the endemic areas due simply to the parasitic organs squeezing,tissue atrophy and necrosis etc,and also,the infection of livestock causes serious economic losses.This study to 1)establish the model of E.g infection combined with DSS according to the theory of "hygiene hypothesis",and observe the influence of parasitic infection on the occurrence and development of enteritis;2)extract and purify thesecretory Antigen B and recombined Antigen B,and analyses the possible mechanism of AgB’s influence on RAW264.7 cells and mouse peritoneal macrophages;3)evaluate the role of AgB against IBD in the mice model of secondary infection and the model of enteritis induced by DSS intervened and to analyze the possible mechanism of the protective effect of AgB on enteritis,so as to provide a theoretical basis for the prevention and treatment of parasitic secretory antigens in related diseases;4)analyzed the changes of intestinal microflora macrogenomics in mice infected by E.g infection or AgB and its effect on immune status of organism and the intestinal microorganism in enteritis model.Methods:1)The IBD model induced by DSS was established on the basis of mice model infected with E.g.The inflammation degree of IBD and the intervention effect of E.g infection were evaluated by body weight,disease activity index(DAI)and pathological score.Peritoneal lavage fluid cells of E.g-infected mice were injected into DSS model mice to evaluate the effect of ascites cells in mice infected with E.g on IBD symptoms;2)nAgB was obtained from the cystic fluid of fresh sheep hepatic cysts infected with E.g.rAgB antigen was induced,expressed and purified from Escherichia coli;RAW264.7 cells and peritoneal macrophages of mice were used for in vitro experiments.Cell proliferation and differentiation indexes were observed after AgB treatment.The effect of AgB on LPS-induced cell injury and the expression of inflammatory factors in M1 cells were analyzed by LPS intervention.Immunofluorescence method was used for identifying the location of AgB in the macrophage membrane.3)After AgB’s intraperitoneal injection,DSS was used to induce enteritis,the inflammation degree of IBD model and the intervention effect of AgB were evaluated by body weight,DAI index and pathological score index,and the indicators of M1 and M2 macrophages in intestinal lamina propria lymphocyte were analyzed;4)The fresh feces of mice were collected from the models of secondary E.g infection,and the genomic DNA from the feces was sequenced by MiSeq 300 PE sequencing platform,then the flora evaluation,flora difference,microbiome function prediction were carried out by the bioinformatics and statistical analysis of 16 S rRNA gene,then the immunological correlation analysis were carried out to explore the effect of E.g infection on the intestinal microflora of mice,then the IBD model of AgB intervention was established,and the effects of AgB intervention on the intestinal microbial structure and functions were observed and analyzed with the above methods.Results:1)The model of IBD was successfully established,the results showed that the body weight of mice induced by DSS was significantly reduced,DAI physiological score increased significantly,and HE staining of colonic tissue sections after dissection showed that inflammatory cell infiltration and tissue damage in the upper part of the colon were significantlt higher than those in the control mice.However,E.g infection combined with DSS model mice showed low inflammation and tissue damage,compared with DSS model group,and the intervention of E.g-infected mouse peritoneal lavage fluid cells on IBD model mice resulted in significant improvement in body weight;2)High purity native and recombined AgB was obtained,in vitro experiments showed that AgB reduced LPS-induced cell damage and the expression of inflammatory factors in M1 cells although didn’t affect cell’s proliferation or differentiation,and AgB could be anchored the surface of cell membrane by confocal image analysis;3)The results showed that the indexes of “AgB+DSS”group were improved compared with “DSS”group,such as the body weight,DAI index,pathological scores,and the proportion of macrophages in abdominal cavity et al.4)Intestinal microflora analysis showed that the main and core microflora in the two groups infected or noninfected with E.g had difference in the level of genus of intestinal bacteria.Lefse analysis revealed that E.g infection increased two intestinal microflora genera: Eisenbergiella and Parabacteroides.The functional prediction of microbial community in E.g infected group and non-infected group showed that seven pathways of intestinal microbial community in E.g infected group changed.The results of association between intestinal macrogenomics and homologous antibodies against HCF showed that there was a strong correlation between intestinal bacterial composition and IgG antibody level of anti-HCF antigen in E.g infected group,and AgB intervention also changed the composition and characteristics of intestinal macrogenomics in IBD mice.There were ecological differences among the control group samples.At phylum level,there was no significant difference in the abundance of bacteria among the groups.However,AgB injection could reverse the increasing trend of Proteobacteria in IBD state and the decreasing trend of Bacteroidetes.AgB injection at phylum level also induced sphingomycetes in enteritis group samples.(Sphingobacteriia and Alphaproteobacteria increased significantly.The functional prediction of intestinal community showed that the four metabolic pathways and P53 signal transduction pathways of bacteria also changed significantly.Conclusion:1)The model of E.g infection combined with IBD was successfully established,and enteritis symptom was significantly inhibited after E.g infection,suggested that E.g infection can protect intestinal inflammation and M2 macrophages in the abdominal cavity may be involved in the process;2)High purity natural and recombinant AgB was obtained,and vitro experiments showed that AgB did not affect cell proliferation and differentiation but reduced LPS-induced cell damage and the expression of inflammatory factors in M1 cells,and AgB could be anchored surface of cell membrane;3)The results showed that index of “AgB+DSS”group were improved compared with “DSS” group such as the body weight,DAI index and pathological score,and the proportion of macrophages in abdominal cavity and lymphocyte M1 and M2 macrophages in proprial layer of colon were changed;4)The species richness of intestinal flora in mice infected with E.g.increased,possibly by some rare bacteria with very low abundance.Whether these rare bacteria are specific changes caused by E.g.infection and the intestinal or immune status of mice need to be further verified and studied.LEfSe analysis shows that Eisenbergiella and Parabacteroides are rare bacteria.Characteroides increased significantly in E.g infected mice,suggesting that they can be used as biomarkers for predicting E.g infection.Functional prediction showed that the intestinal microflora of mice infected with E.g had seven pathways,and the composition of the major genera was positively correlated with IgG1 and IgG2 b.Further studies are needed to understand the possible mechanism of intestinal microflora changes after helminth infection and to identify changes in intestinal microflora in CE patients.AgB intervention had an important effect on the composition and characteristics of intestinal macrogenomics in IBD mice: AgB intervention changed the ecological characteristics of intestinal flora in enteritis and changed the abundance of intestinal flora,metabolic pathway and P53 signal transduction pathway at different levels of class,order,family and genus.Combined with the results of thesecond part of this project,it is shown that the change of immune status caused by AgB intervention may affect the macrogenomics of intestinal colonies,thus resulting in the protective effect of AgB on enteritis. |
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