Trinitrobenzene Sulfonic Acid (TNBS) Induced Irritable Bowel Syndrome(IBS) And The Mechanisms Of Clostridum Butyricum Alleviated Intestinal Permeability And Immune Dysfunction

BackgroundIrritable bowel syndrome (IBS) constitutes a functional gastrointestinaldisorders characterized by abdominal pain/discomfort, meteorism with abdominaldistension and alterations in bowel movements, which cannot be explained bystructural or biochemical abnormalities, sharing a poorly definedpathophysiology. Irritable bowel syndrome (IBS) is a common gastrointestinaldisorder characterized by abdominal pain and alterations in bowel habits affecting.statistically, prevalence rate was5%-25%of people worldwide, Europe and otherdeveloped countries were10%-15%, and the incidence rate range from15%to20%,while incidence rate of5%to10%, an upward trend in recent years in Asia, theprevalence of IBS are quite different in various regions, espically in China. Currently,genes, psychosocial factors, dysfunction of the brain–gut axis, gastrointestinalinfection, gastrointestinal motility disorder and visceralhypersensitivity are thought toplay crucial roles in symptom generation, but the pathogenic mechanism of IBSremains incompletely understood. One of the most important reasons is the difficultyinvolved in developing a validated IBS animal model. Partly intestinal CD4+T cells were thought to be involved in the pathogenesis of IBS And probiotics canalleviate IBS symptoms in clinic.There is increasingly convincing evidence supporting the participation of the gutmicroenvironment in the pathophysiology of irritable bowel syndrome (IBS). Studiesparticularly suggest an interplay between luminal factors (eg, foods and bacteriaresiding in the intestine), the epithelial barrier, and the mucosal immune system.Decreased expression and structural rearrangement of tight junction proteins in thesmall bowel and colon leading to increased intestinal permeability have beenobserved.AimsTo investigate the role of intestinal immune dysfunction in the pathogenesis ofirritable bowel syndrome(IBS) induced by intrarectally trinitrobenzene sulfonicacid(TNBS). and phenotype and function of CD4+T cells changed in the development ofIBS mouse model. The effects of Clostridum butyricum on the regulation of low-gradeinflammatory, intestinal immune disorders and intestinal permeability changes in thepathogenesis of irritable bowel syndrome(IBS).Methods(1) A total of80male6-week-old C57BL/6mice were randomly divided into threegroups, including the experimental group(n=20), the control group(n=10) and theClostridum butyricum group(n=20), the standard strains group (n=20), untreatedmouse(n=10). The TNBS-IBS model were induced by intrarectally introducing asingular dose of0.2ml trinitrobenzene sulfonic acid (TNBS)(2.5mg/mouse in30%EtOH). The mice in control group were intrarectally perfused with normal saline enema(200μl). Two hours before the perfusion of sodium butyrate into colon, the mice inClostridum butyricum group(n=20) were given Clostridum butyricum500μl(viable cell concentration of1×109CFU/ml) by oral gavage once a day for six days.At the same time, the standard strains group (n=20) using standard strains (E. coli)contrast evaluation the role of probiotics of Clostridum butyricum.10untreated mousewere used as na ve mice. The colorectal distention test(CRD) was carried out forevaluation of clinical parameters. H&E staining of intestinal tissue section wasperformed for histopathological assessment of colonic mucosal inflammation.Intestinal intraepithelial lymphocytes (IELs) and lamina propria mononuclearcells(LPMCs) were isolated and analyzed by Flow cytometry to evaluate thecorrelation between IBS and intestinal immune dysfunction/abnormal activation of intestinal immune cells in mouse model of C-IBS, and to assess the regulatory effectsof Clostridum butyricum on the intestinal immune disorder.(2) The TNBS-IBS model were induced by intrarectally introducing a singulardose of0.2ml trinitrobenzene sulfonic acid (TNBS)(2.5mg/mouse in30%EtOH) inCD4-KO mice (C57BL/J6background). And then mice were treated with TNBStogether with or without gavage-feding Clostridum butyricum. The wildtype(C57BL/J6) mice in control group were intrarectally perfused with same volumeof TNBS enema (200μl). On the day6and day24, the perception of pain sensationwas assessed by the colorectal distension (CRD).CD4+/8+T cells in peripheral bloodmononuclear cells (PBMCs) of blood and macrophages and memory T cells in thelamina propria were analyzed by flow cytometry. Adoptive transfer of CD4+T cellsto CD4-KO mice IBS model which had been treated with Clostridum butyricum,evaluating its immune disorders in the situation.Results(3) CD4+T cells and intestinal lamina propria dendritic cells (LPDCs) wereisolated and purified by intestine digestion and magnetic label-based technique.Surface markers were analyzed by flow cytometry. Endocytic activity, mixedlymphocyte reaction (MLR) and chemotaxis were studied. Cytokines and proteinsproduction of the CD4+T cells cocultured with LPDCs, lasting for three days,weredetermined.(4) A total of40male6-week-old C57BL/6mice were randomly divided intofour groups, including the experimental group(n=10), the control group(n=10) and theClostridum butyricum group(n=10), the standard strains group (n=10), untreatedmouse(n=10). The TNBS-IBS model were induced by intrarectally introducing asingular dose of0.2ml trinitrobenzene sulfonic acid (TNBS)(2.5mg/mouse in30%EtOH). The mice in control group were intrarectally perfused with normal salineenema (200μl). Two hours before the perfusion of sodium butyrate into colon, themice in Clostridum butyricum group(n=20) were given Clostridum butyricum500μl (viable cell concentration of1×109CFU/ml) by oral gavage once a day for six days.At the same time, the standard strains group (n=10) using standard strains (E. coli)contrast evaluation the role of probiotics of Clostridum butyricum.10untreatedmouse were used as na ve mice. on twenty four days,(1) Detecting physiologicalindicators of intestinal pathological damagephysiological, pathological intestinalinjury and visceral sensitivity;(2)Detecting mouse D-lactic acid (D-LA), mousediamine oxidase (DAO) and myeloid catalase (MPO) levels;(3) Western Bolt Technical Analysis intestinal epithelial cells (IEC) cell tight junction protein (TJ)(occludin-1, claudin-1, ZOL-1) protein levels. Evaluation of Clostridum butyricum (C.butyricum) on the key role of intestinal permeability.ResultsRectal Instillation of TNBS is a stable useful IBS animal model. Comparedwith control group, the mice in experimental group showed a significant change inphysiological parameters, histological structure of colon, inflammatory cellsinfiltration and low-grade inflammatory state. There was a significant increase inscores of CRD and a decrease in lowest sensory threshold (t=8.926and t=6.103,bothP＜0.001);(2)There was a decrease in the numbers of DCs in IELs (t=2.878andt=3.086, both P＜0.05), but an increase in the numbers of macrophage (t=3.191,P＜0.05) and nearly unchanged memory T cells in mice with IBS as compared withcontrol group;(3)DCs were decreased (t=2.880and t=2.664, both P＜0.05), butmemory T cells were increased (t=3.732and2.682, P＜0.01and P＜0.05) in theLPMCs of mice in experimental group;(4)There was no significant difference in thephysiological index between control group and Clostridum butyricum group. Levels ofmemory T cells, macrophages and DCs in the IELs were close to the normal level (6d, t=1.103,0.0243,0.418, all P>0.05), and levels of macrophages and DCs in the LPMCsof Clostridum butyricum group were also similar to control group (6d, t=0.782,0.347,both P>0.05);(5)Compared with experimental group, the level of memory T cells inLPMCs of mice treated with Clostridum butyricum was dramatically declined (6d, t=2.346, P=0.0470, P <0.05), however, which was still higher than control group (6d, t=2.233, P=0.0476, P <0.05). The intestinal immune function was restored to normallevel with Clostridum butyricum intervention.Clostridum butyricum restores intestinal immune dysfunction of irritablebowel syndrome mouse model. Mice treated with TNBS showed a significantincrease in scores of CRD and decrease in pain threshold were recorded; thefrequency of CD4+and8+T cells were significantly increased in the PBMCs of theblood in TNBS-group of wild type mouse, and the CD8+T cells increased more thanwide type mouse model; the frequency of CD4+and8+T cells were significantlyincreased in the lamina propria mononuclear cells(LPMCs) of the colon inTNBS-group of wild type mouse, and the CD8+T cells increased more than widetype mouse model; the frequency of CD4+T cells were significantly increased in theLPMCs of the colon in TNBS-group wide type mouse as well as the CD8+T cells were significantly increased in CD4-KO IBS mouse; Adoptive transfer of CD4+Tcells reduced the immune reaction of colon intestinal low-grade inflammationintensity.Clostridum butyricum effective regulates the pathogenic of CD4+T cells ofirritable bowel syndrome.(1) Enhanced ability of endocytic activity and decreasedabilities to attract and stimulate na ve CD4+T cells proliferation were in TNBS group;(2) Cocultured LPDCs with CD4+Tcells showed a predominant Th1, Th17responsein the IBS stage, and more important roles of Th2and Treg responses in the IBS stage,which treated with Clostridum butyricum;(3) Cytokines of TNF-α, IFN-γ, IL-1β,IL-4,IL-13, IL-6, IL-17, IL-10and TGF-β and the same proteins production of theCD4+T cells were almost likely with CD4+T cells subsets change.Clostridum butyricum remarkable alleviates of the abnormal increasedIntestinal permeability on irritable bowel syndrome in mouse model. Comparedwith control group,(1)TNBS experimental mice showed physiologicalcharacteristics significant changed(all P<0.05). the mice in experimental groupshowed a significant change in low-grade inflammatory state in colon. There TNBS asignificant increase in scores of CRD and a decrease in lowest sensory threshold (allP<0.05). After intragastric intervention with Clostridum butyricum and E. coli, thevisceral hypersensitivity were significantly improved (all P<0.05), colon mucosalinjury TNBS significantly improved and the number of infiltrating inflammatorycells were significantly reduced(P<0.05);(2)the TNBS group’s serum D-LA, DAO,MPO levels were significantly increased (all P<0.05). After the intervention withClostridum butyricum and E. coli, the serum D-LA, DAO, MPO levels weresignificantly lower than TNBS group (all P<0.05). Clostridum butyricum group werebetter than E. coli group in D-LA, DAO degree(both P<0.05);(3)TNBS group’soccludin-1, ZOL-1protein levels were significantly decreased(both P<0.05), whenintervention with Clostridum butyricum and E. coli, the occludin-1, ZOL-1levelswere significantly increased(both P<0.05), and Clostridum butyricum group werebetter than E. coli group (both P<0.05). The claudin-1protein level of TNBS notchanged significantly(all P＞0.05).Conclusions1. Rectal Instillation of TNBS could induce the IBS-like symptoms in mice,was a stable useful IBS animal model.2. Discovered and comfirmed intestinal increased intestinal mucosal permeability complex integrity associated with activation and differentiation ofCD4+T cell subtypes, dendritic cells (DCs), macrophage and memory T cellsabnormal distribution and activation, which were important pathophysiologicalmechanism of IBS.3. This study adds novel information to demonstrate Clostridum butyricumcan effectively anti-inflammatory, reduce intestinal mucosal permeability,alleviate immune dysfunction, decrease visceral hypersensitivity and recovery ofgastrointestinal function.