Probiotics Inhibit Inflammatory Reaction Of Intestine In Inflammatory Bowel Disease

Part Ⅰ:Probiotics inhibit TNF-α-induced interleukin-8 secretion of HT29 cellsBACKGROUND 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 HT29 cells which have basically the same biological properties of normal colonic epithelia with some probiotics, and stimulated the cells by TNF-α to intimate intestinal inflammatory condition, in order to study the effect of probiotics on proinflammatory cytokines secretion of epithelia in intestinalinflammation.METHODS: Colonic adenocarcinoma HT29 cells were cultured and divided into four groups: ControK TNF- a (group T in short)> Bifidobacterium (group B) -. Lactobacillus (group L) . B. Longum and L. bulgaricus were suspended in culture medium with the concentration of 1xlO8 cfu/ml and added into 24 wells respectively. 1 hour later TNF- a (lOng/ml) was added into each well of three groups as T> B> L. The IL-8 expression of each group was measured by ELISA and RT-PCR after 3 hours, nuclear factor- k B (NF- k B) P65 was also examined by Western blotting.RESULTS: There was less interleukin-8 secretion of HT29 cells when preincubated with B. Longum or L. bulgaricus compared with group T. Less NF- k B P65 appears in nucleus in group B and L compared with group T, as detected by Western blotting.CONCLUSION: Probiotics suppress interleukin-8 secretion of HT29 cell line when stimulated by proinflammatory cytokine, which is most likely mediated by inhibited NF- k. B activation.Part II:The effect of Bifidobacterium on murine experimental colitisBACKGROUND AND AIMS: The etiology of inflammatory boweldisease(IBD) is still unknown, but more and more investigations showed that gut microfiora plays important role in pathogenesis of IBD. Microflora in IBD gut becomes aberrant, with normal microflora 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 dextran sulfate sodium(DSS)-induced colitis in mice which has the same pathological property as human UC, and observed the effects on colonic inflammation with Bifidobacterium supplementation, to study the therapeutic effect of bifidobacterium and its possible mechanism.METHODS: Mice were randomly divided into four groups: ControK DSS^ SASP, and Bifidobacterium(Bif in short). Mice of groups DSS, SASP, and Bif were fed with 5% DSS(w/v) solution for 7 days to induce colitis, mice of Control just drink distilled solution, and disease activity index (DAI) was calculated every day. Mice of SASP were fed with SASP every day during inducing colitis, and mice of Bif were given Bifidobacterium by oral gavage from 7 days before the experiment to the end of experiment. The expression of TNF- a ^ NF- k. B P65, Fas and MPO in inflamed colon of each group mice was measured at the end of experiment.RESULTS: The mice in group SASP and Bif showed a lower DAI than those in group DSS since the forth day to the end of experiment. There were lower level of TNF- a and MPO in murine inflammatory colon, lower NF- k B P65 expression in nuclei of inflammatory cells, lower Fas expression in colonic epithelia of group SASP and Bif comparing with group DSS at the end ofexperiment.CONCLUSION: Treatment with bifidobacterium has beneficial effect on murine experimental colitis, the mechanism may be involved in following respects: Bifidobacterium inhibits proinflammatory cytokine secretion and NF-k B activation in inflammatory cells, decreases colonic inflammatory reaction, downregulates Fas expression in colonic epithelia of murine inflamed colon, alleviates inflammatory damage of colonic epithelia and protects the integrity of intestinal mucosal barrier.Part HI:probiotics modulate tissues and cells function ofinflamed intestinal mucosa in active ulcerativecolitis patientsBACKGROUND 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 indigenousmicroflora, 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 ulcerative colitis patients with bifidobacterium and lactobacillus, and measured the proinflammatory cytokines from supematants to find out the modulation effects of probiotics on inflammatory colonic specimens and investigated its possible mechanism.METHODS: Colonic specimens from active ulcerative colitis patients were collected by endoscopy, and divided randomly into three groups: Control -> Probiotics(Prob)> Dexamethason(DEX). All specimens were cultured by culture medium, and those of group Prob were cocultured together with 1X 106cfu/ml bifidobacterium and lactobacillus, those of DEX were treated by 15 u mol/ml dexamethason. 24 hours later, TNF- a ., IL-8 in supernatant of each group were measured by ELISA; The specimens were fixed by paraffin, and expression of NF- k. B P65 -. Fas in tissues was also studied by immunohistochemical staining.RESULTS: The concentrations of TNF- a ? IL-8 in supematants of Prob and DEX were lower than that of Control, and the lowest was in DEX group. The expression of NF- k B in nuclei of inflammatory cells in Prob and DEX was less than that of Control. The expression of Fas in intestinal epithelia was less than Control too.CONCLUSION: When cocultured with colonic specimens from ulcerative colitis patients, Probiotics can downregulate inflammatory cytokineproduction in those tissues and decrease epithelium cell damage. The possible mechanism may be related to below: Probiotics inhibit NF- k B activation in inflammatory cells and downregulate Fas expression of inflammed intestinal epithelia.