| Part I: The Study on effects and mechanism of intestinal epithelial cells of Bifidobacteria BB12 in inflammatory stateBACKGROUND AND AIMS: Intestinal epithelium is an important component of gut mucosal barrier, and participates in mucosal innate immunity. The normal microflora colonizes on the mucosal surface of intestinal tract, and modulates intestinal epithelia physiological function. In inflammatory condition, gut microflora becomes aberrant and induces physiological function disorder of intestinal epithelia, and epithelia secrete a lot of cytokines which can amplify dysregulated immune reaction. Supplement with probiotics experimentally could recover the microbial environment by resuming balance microflora, and inhibit intestinal abnormal immune reactivity. In this experiment, we cocultured Caco-2 cells which have basically the same biological properties of normal colonic epithelia with B.lactis BB12, and stimulated the cells by Salmonella enteritidis and inflammatory cytocine respectively to intimate intestinal inflammatory condition, in order to study the effect of BB12 on proinflammatory cytokines secretion of epithelia in intestinal inflammation. METHODS: In vitro, Colonic adenocarcinoma Caco-2 cells were cocultured with BB12, and at same time cells were stimulated the cells by Salmonella enteritidis and inflammatory cytocine respectively. The IL-8 expression of each group was measured by RNA blot,Real-time PCR,RT-PCR and ELISA. Immunofluorescence microscopy and EMSA for the NF-κB P65 and in vitro kinase(IKK) assay . For in vivo, Salmonella enteritidis was fed to rat with BB12 or without BB12.RESULTS: In vitro, BB12 inhibited the secretion of IL-8 induced by by Salmonella enteritidis and inflammatory cytokine and attenuated nuclear NF-κBp65. BB12 also reduced IκB phosphorylation and blocked IKK activity. In vivo, BB12 preserved intestinal structure and attenuate gut inflammation.CONCLUSION: BB12 suppress interleukin-8 secretion of Caco-2 cell line when stimulated by Salmonella enteritidis and proinflammatory cytokine, which is most likely mediated by inhibited blocks NF-κB signaling pathway by targeting IKK complex in Caco-2 cells .Part II: The Study on Bifidobacteria BB12 in Prevention and Treatment of a Rat Model of Crohn's disease.BACKGROUND AND AIMS: The etiology of inflammatory bowel disease(IBD) is still unknown, but more and more investigations showed that gut microflora plays important role in pathogenesis of IBD. Microflora in IBD gut becomes aberrant, with normal micrflora decreased, harmful and potential harmful bacteria increased. As have been reported, there has close relationship between gut aberrant microflora and mucosal immune function disorder, and modification of intestinal microflora may have therapeutic effect on IBD. In this experiment, we used TNBS-induced colitis in rats which has the same pathological property as human CD, and observed the effects on colonic inflammation with Bifidobacterium supplementation, to study the therapeutic effect of bifrdobacterium and its possible mechanism.METHODS: Mice were randomly divided into five groups: Normal control, positive control, prevention group, treatment group, and 5-ASA. Rats of 5-ASA were fed with 5-ASA every day during inducing colitis, and Rats of BB12 were given BB12 by oral gavage from 15 days before the experiment to the end of experiment. The expression of TNF-α,NF-κB P65 and MPO in inflamed colon of each group mice was measured at the end of experiment.RESULTS: BB12 significantly reduced the severity of TNBS-induced murine colitis as assessed by clinical disease activity score, gross score and histology. The concentration of TNFα,LDH,AKPand MPO in prevention group,treatment group and 5-ASA group is lower than in positive control group . Furthermore, tissue upregulation of IκB and IKK phosphorylation induced by TNBS was attenuated by BB12.CONCLUSION: Treatment with bifidobacterium BB12 has beneficial effect on experimental colitis, the mechanism may be involved in following respects: Bifidobacterium inhibits proinflammatory cytokine secretion and NF-κB activation in inflammatory cells, decreases colonic inflammatory reaction.Part III: Biffidobacterium BB12 modulate tissues and cells function of inflamed intestinal mucosa in active Crohn's disease patients BACKGROUND AND AIMS: There have at least 500 different microbial species in gut, those species have different biological properities and functions. The normal microflora may interact with intestinal epithelia and mucosal immune system. However, the interaction is disordered and inflammation accompanied by imbalance in the intestinal microflora. Supplement with probiotics may stabilize and balance the indigenous microflora, and normalize the host-microbe interaction. In the former experiments, we have found probiotics can downregulate inflammatory cytokines secretion of intestinal epithelia under inflammatory condition, and alleviate intestinal mucosal inflammation of murine experiment colitis. In the experiment, we cocultured colonic specimens from Crohn's disease patients with Bifidobacteria BB12, and measured the proinflammatory cytokines from supernatants to find out the modulation effects of probiotics on inflammatory colonic specimens and investigated its possible mechanism.METHODS: Colonic specimens from active Crohn's disease patients were collected by endoscopy, and divided randomly into three groups: CD,BB12 and 5-ASA. All specimens were cultured by culture medium. 24 hours later, TNF-α,IL-8,LDH and AKP in supernatant of each group were measured by ELISA; The specimens were fixed by paraffin, and expression of NF-κB P65 was studied by imununohistochemical staining.RESULTS: The concentrations of T NF-α,IL-8,LDH and AKP in supematants of BB12 and 5-ASA were lower than that of CD control group, and the lowest was in 5-ASA group. The expression of NF-κB P65 in nuclei of inflammatory cells in BB12 and 5-ASA was less than that of CD control group. CONCLUSION: When cocultured with colonic specimens from CD patients, BB12 can downregulate inflammatory cytokine production in those tissues and decrease epithelium cell damage. The possible mechanism may be related to below: Bifidabateria BB12 inhibit NF-κB activation in inflammatory cells...
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