The Research In Probiotic Effects Of A Novel Strain Of Bacteroides Fragilis And Its Polysacchride A

Background&AimsPreviously we isolated a novel strain of Bacteroides fragilis(B.fragilis)SK08,and obtained polysaccharide A with up to 99%purity from it.Although the approval has been obtained by national institutions,its probiotic effects should be explored unceasingly.This study comprises of two parts,the first of which is focus on the preventive effects of B.fragilis SK08 on Clostridium difficile infection(CDI),while the second of which is focus on the effects of TP2 on colitis associated colorectal cancer(CAC).First Part:SK08 Prevents Clostridium difficile Infection in a Mouse Model by Restoring Gut Barrier and Microbiome RegulationMaterials and methodsCDI mice were treated with 1×107 cfu or 1×108 cfu B.fragilis SK08 at Day 0.The body weight,diarrhea and death were monitored.The cecum and colon were analysed by HE staining,immunohistochemical analysis of MUC-2 and ZO-1 proteins,and Western blot analysis of apoptosis proteins.The gut microbiota in cecal content were analysed by 16S rRNA sequencing.In vitro experiments were performed to confirmed the inhibition of B.fragilis SK08 on growth and toxin release of C.difficile,adhesion,cell apoptosis and gut barrier dysfunction induced by C.difficile.Results:CDI model mice prophylactically treated with B.fragilis SK08 exhibited higher survival rate and improved clinical manifestations.Histopathological analysis of colon and cecum tissue samples revealed an intact gut barrier with strong ZO-1 and Muc-2 expression.Western blot results suggested a higher expression of anti-apoptosiss reaction in B.fragilis treated mice.The richer bacterial diversity and the increased relative abundance of gut microbiota related to bile acid metabolism and gut barrier protection was positively correlated with B.fragilis treatment.In vitro experiments showed that the supernatant of B.fragilis significantly inhibited the growth and toxin release of C.difficile,and viable B.fragilis inhibited C.difficile adherence,and attenuated the decrease in CDI-induced transepithelial electrical resistance,ZO-1 and MUC-2 loss,and apoptosis.Conclusions:In summary,B.fragilis exerts protective effects on a CDI mouse model by modulating gut microbiota and alleviating barrier destruction,thereby relieving epithelial stress and pathogenic colitis triggered by C.difficile.This study provides an alternative preventative measure for CDI and lays the foundations for further investigations of the relationships among opportunistic pathogens,commensal microbiota,and the gut barrier.Second Part:TP2 Prevent Colitis Associated Colorectal Cancer through Regulating Gut Microbita and Tumor Associated Macrophage PhenotypeMaterials and methodsDetected the cytotoxicity of TP2.Treated the mice with TP2 for 80 days and monitored the basic conditions,colon,serum cytokines and constitution of gut microbiota.Prophylactically treated CAC mice with TP2 and monitored the occurrence of diarrhea and bloody stool,the number of tumor.Tested the concentrations of cytokines in serum,and sequenced the gut microbiota.Detected the ratios of infiltrated CD11b+F4/80+,CD11b+Ly6g+and CD8+T cells.Constructed the in vitro TAM cell model and detected the transcriptional changes induced by TP2 of a few genes by RT-PCR method.Results:TP2 did not display influences on the adhesion,viability and proliferation of colon cells in vitro.TP2 did not induce diarrhea,weight loss and death during treatment.The concentration of cytokines were not increased and observed species and relative abundance of gut microbiota accounting for metabolism metabolism were increased in TP2 treated mice.Improved the occurrence rate of diarrhea and bloody stool and reduced the number of tumors were observed in TP2 treated CAC mice.In addition,TP2 treatment significantly inhibited the release of IL-6,TNF-α,and decreased the infiltrating ratios of CD 11 b+F4/80+ and CD 11 b+Ly6g+cells,but significantly increased CD8+T cells in CAC mice.In vitro results suggested that TP2 inhibited transcriptional levels of several genes in TAM in virto model,and the cell proliferation of colon cells were also inhibited.In sum,TP2 invented by our team is safe both to cells and to mice.Long term intake of TP2 regulates the gut microbiota in mice.Conclusions:TP2 significantly improves the symptoms of colitis and inhibited the tumorigenesis in CAC mouse model,properly in correlation with regulating mice gut microbiota and tumor micro-environment.TP2 has the ability to inhibit tumor cells in vitro by regulating TAM phenotype.