Relieving Effect And Mechanism Of Bifidobacterium Bifidum On TBNS-induced Colitis

Inflammatory bowel disease(IBD),which includes Crohn’s disease(CD)and Ulcerative colitis(UC),is a relatively common chronic digestive tract disease.It is mainly caused by the complex interaction of immune disorders,genetic predisposition and intestinal microbiota.Over the past decade,IBD has become a global public health challenge,putting a great burden on people’s health and quality of life.Conventional immunotherapy does not maintain long-term remission of IBD and has many side effects.Regulatory approaches targeting the microbiome,probiotic agents such as Bifidobacterium,appear to be novel approaches to curing IBD.However,the efficacy of Bifidobacterium in alleviating IBD appears to be strain specific,and the functional genes,microbial characteristics and metabolic activity of the strains may be the important factors affecting the efficacy of Bifidobacterium.There is still a lack of standard screening methods for effective strains of IBD treatment.Moreover,the therapeutic modalities and underlying molecular mechanisms of Bifidobacterium remain unknown.In this study,several strains of Bifidobacterium bifidum(Type species of Bifidobacterium)were selected,and2,4,6-trinitro-benzene acid(TNBS)-induced colitis in mice were used as models to investigate the potential efficacy and mechanism of Bifidobacterium in alleviating IBD.The microbiological characteristics,functional genes and immune stimulating ability of B.bifidum strains with different anti-colitis effects were analyzed.Then the activities of different components of B.bifidum strains were evaluated,and finally the material basis of B.bifidum in alleviating TNBS-induced colitis was determined.In-vivo validation was performed.The main results of this paper are as follows.First,three mouse models of acute gut injury,acute colitis and chronic colitis induced by TNBS were established.Nine strains of B.bifidum were used for intervention,and the alleviating effect of colitis was evaluated by mortality,disease activity index,colon pathology,intestinal fibrosis,the level of inflammatory factor,intestinal barrier,fecal metabolite level and immunity index.The results showed that strains of B.bifidum had different alleviating effects on TNBS-induced colitis.Strains JSNJJNM2,CJ238 and FJSWX19M5 could significantly alleviate the symptoms of weight loss,DAI index,colon injury and increased levels of inflammatory factors in colitis mice,and the anti-colitis effect was better.Strains M203F02M632,FXJWS17M4 and FBJ1M4 had no significant alleviating effect on the above indexes.The relationship between the alleviating effect of specific B.bifidum on TNBS-induced colitis and intestinal microbiota was explored through the antibiotic-induced gut microbes depleted mouse model,and the effect of B.bifidum on intestinal microbiota of TNBS-induced colitis mice was evaluated,and then the effect of strain activity,dose and prevention methods on the alleviating effect of B.bifidum on TNBS-induced colitis was studied.The results showed that oral adminstration of B.bifidum has no significant change in the structure of gut microbiota in mice with acute colitis.When the intestinal flora was depleted,some B.bifidum strains still had a relief effect on acute colitis.High dose(10~9 CFU/d)of heat-killed B.bifidum showed protective effect on TNBS-induced colitis mice.The prophylactic effect of B.bifidum on acute colitis is superior to its therapeutic effect.Then,functional genes,basic characteristics of microbiology,and immunostimulation ability of B.bifidum with different anti-colitis abilities were determined by comparison of genomes and co-culture of mesenteric lymph node(MLN)cells.The correlation analysis of physiological characteristics and functional genes of B.bifidum with its anti-colitis indexes(colon length and spleen index)showed that the effect of B.bifidum on TNBS-induced colitis was not related to its in-vitro proliferation ability,but to its adhesion and immuno-stimulatory activity(the ability to induce MLN to produce IL-10,IL-17,and IL-12).Compared with the ineffective strains(M203F02M632,FXJWS17M4 and FBJ1M4),B.bifidum strains with significant anti-colitis ability(CJ238,FJSWX19M5)contained 13 gene clusters that could encode Sortase,LPXTG cell wall anchor motif,etc.Finally,different components of B.bifidum were isolated,and their immune activity was evaluated using in-vitro MLN and bone marrow-derived dendritic cells(BMDC)cell models to determine the active components of B.bifidum associated with its anti-colitis effects.The most immunoactive component was selected to verify the relieving effect of colitis in normal and germ-free mouse models.The results showed that compared with peptidoglycan,capsular polysaccharide and total membrane proteins,lipoteichoic acid(LTA)had stronger immunostimulatory capacity to induce IL-10 secretion in MLN cells,which was dose-dependent within a certain concentration range and dependent on toll-like receptors(TLR)-2rather than TLR4 receptor signaling.The immune stimulation ability of different strains of LTA was related to its anti-colitis ability.LTA derived from FJSWX19M5 showed significant protective effects on both WT and GF mice with TNBS-induced acute colitis and also could promote the proliferation of spleen lymphocytes and delayed type hypersensitivity(DTH)in normal mice.