Study Of The Effect And Mechanisms Of CLA Producing Bifidobacterium On Alleviation Of Colitis

Inflammatory bowel disease(IBD)comprises ulcerative colitis(UC)and Crohn's disease(CD);the main characteristic of which is intestinal mucosal inflammation,severe clinical responses and frequent recurrences.However,prolonged use of traditional medicine(budenisonide,5-aminosalicylic acid and hydrocortisone)may cause side-effects such as osteoporosis,diabetes and hypertension,thus,affecting the efficiency of treatment.New therapies such as monoclonal anti-TNF-?,probiotics,prebiotics and unsaturated fatty acids are used for IBD in place of traditional medicines,which can modulate the gut microbiota and interfere with host immune response.Although the role of probiotics in regulating intestinal barrier and alleviating colitis has been confirmed by a large number of studies,there are limited studies on the material basis,dose-effect relationship,regulatory mechanism and clinical effects of probiotics in alleviating colitis.In this context,this subject compared the alleviating effect of conjugated linoleic acid(CLA)-producing bifidobacteria on UC,investigated the material basis and target,and explored the mechanism of alleviating colitis as well ass the dose-effect relationship from the aspects of intestinal immunity and mechanical barrier.The main results are as follows:Firstly,the difference of CLA-producing bifidobacteria in alleviating colitis and the relationship between CLA and their alleviating effect on colitis were studied.A mouse model of colitis was established by DSS.A total of 15 CLA-producing Bifidobacterium(B.breve,B.longum and B.pseudocatenulatum)were administered to colitis mice by gavage.The effective strains were screened based on the pathological characteristics,and the differences were compared to analyze the correlation between the effect of alleviating colitis and CLA producing.The results showed that B.breve CCFM683,B.breve BJCP1M6,B.longum CCFM681,B.pseudocatenulatum MY40C and B.pseudocatenulatum CCFM680 could significantly inhibit the increase of DAI index,prevent colon shortening,improve pathological score and alleviate colitis,while other 10 CLA-producing Bifidobacterium strains had no significant improvement.Furthermore,the colonic CLA concentration was significantly positively correlated with the effect of colitis mitigation,suggesting that CLA may be an important substance of colitis mitigation by these bacteria.Secondly,B.breve CCFM683 was taken as an example to explore the material basis and target of CLA-producing Bifidobacterium to alleviate colitis.The material basis of CLA-producing Bifidobacterium to alleviate colitis was studied through dead and alive bacteria and gene knockout and complement bacteria.The results showed that dead B.breve CCFM683 had no relieving effect on colitis.B.breve CCFM683?bbi,knoctout of linoleate isomrease from B.breve CCFM683,showed no significant improvement in alleviating colitis while the related gene complemented strain,B.breve CCFM683?bbi:bbi,and the wild type,B.breve CCFM683,showed significant relief of colitis,which suggested that CLA was the key material basis to alleviate colitis by B.breve CCFM683.B.breve CCFM683 significantly reduced the effect of alleviating colitis when the mice was treated with PPAR-?inhibitor,which suggested that PPAR-?was an important target of alleviating colitis by B.breve CCFM683.Further,the mechanism of CLA-producer,B.breve CCFM683,alleviating colitis was explored from the intestinal immune barrier and mechanical barrier by cellular,molecular and immune methods.The results showed that B.breve CCFM683 regulated the intestinal immune barrier by producing CLA,up-regulating PPAR-?,inhibiting NF-?B signaling pathway and down-regulating pro-inflammatory cytokines TNF-?and IL-6.Meanwhile,B.breve CCFM683activated the GPR40-ERK-MEK signaling pathway,up-regulated the colonic tight junction protein,regulated the key apoptotic proteins Bad and Bcl-2,alleviated the apoptosis of colon epithelial cells and improved the colon epithelial barrier.Furthermore,B.breve CCFM683promoted the goblet cells to secrete a large amount of MUC2 by CLA production,protect intestinal mucus layer and prevent intestinal microorganism from migrating to colonic epithelium.These results indicated that B.breve CCFM683 could regulate intestinal immunity and mechanical barrier by producing CLA to activate receptor GPR40,inhibit NF-?B signaling pathway after up-regulation of PPAR-?and activate ERK-MEK signaling pathway,respectively.Colitis mice were treated with different concentration of CLA and B.breve CCFM683 to study the dose-effect relationship.The results showed that CLA,a key metabolite,alleviated colitis in a dose-dependent manner by inhibiting pro-inflammatory cytokines and improving the intestinal barrier.Further study on the dose-effect of B.breve CCFM683 showed that both10¹⁰ CFU/day and 10⁹ CFU/day B.breve CCFM683 had significant remission effect on colitis,while less than 10⁸ CFU/day B.breve CCFM683 had no significant remission effect.In addition,the dose of B.breve CCFM683 was positively correlated with the palliative effect of colitis,showing a significant S-shaped dose-response curve.The dose-response curve result showed that B.breve CCFM683 had a significant relieving effect on colitis when the dose of B.breve CCFM683 was more than 10^8.65 CFU/day.