| ObjectiveIrritable bowel syndrome(IBS)is the most commonly diagnosed gastrointestinal condition.In most patients,IBS is a chronic relapsing disease in which symptoms may vary over time.Previous surgery,longer duration of disease,higher somatic scores,and comorbid anxiety and depression all predicted worse out comes,and it significantly reduces health-related quality of life and work productivity.The pathogenesis of IBS is heterogeneous.During the past 40 years,a number of factors that contribute to the pathophysiology of IBS have emerged.Traditionally,the pathogenesis of IBS has focused on abnormalities in motility,visceral sensation,brain gut interaction,and psychosocial distress.Although one or more of these abnormalities are demonstrable in the majority of IBS patients,none can account for symptoms in all of them.More recently,altered gut immune activation,intestinal permeability,and intestinal and colonic microbiome have been identified in some IBS patients.Increased small bowel and colonic permeability has also been observed in patients with IBS-D.The intestinal barrier is a major defense mechanism used to maintain epithelial integrity and to protect the organism from the environment.It serves two critical functions for the survival of the individual: first,it allows nutrient absorption and second,it defends the body from dangerous macromolecule penetration.Disruption of intestinal epithelial barrier function appears to play an important role in the pathogenesis of chronic inflammatory intestinal disorders,such as Irritable bowel syndrome(IBS).Altered gut permeability may increase bacterial load and dietary antigens in the lamina propia leading to mucosal inflammation.Consumption of non-pathogenic bacteria can contribute to intestinal barrier function,and probiotic bacteria have been extensively studied for their involvement in the maintenance of gut barrier.Probiotics are live bacteria that,when consumed in sufficient quantities,confer a health benefit to the host.Prebiotics are nutrients,usually carbohydrates,that encourage the growth of probiotic bacteria.The most robust data have evaluated the role of probiotics for IBS.In a recent meta-analysis including 35 randomized clinical trials,probiotics as a group improved global IBS symptoms(relative risk,0.79;95%CI,0.70-0.89;number needed to treat,7;95%CI,4-12.5),abdominal pain,bloating,and flatulence.Several important mechanisms underlying the antagonistic effects of probiotics on various microorganisms include the following: modification of the gut microbiota,competitive adherence to the mucosa and epithelium,strengthening of the gut epithelial barrier and modulation of the immune system to convey an advantage to the host.In particular,strains belonging to Bifidobacterium and Lactobacillus,which are the predominant and subdominant groups of the gastrointestinal microbiota,respectively,are the most widely used probiotic bacteria and are included in many functional foods and dietary supplements.Clinically,probiotics such as Lactobacilli which isolated from the healthy human intestine has been shown to improve intestinal barrier function in experimental animals and reduce symptoms of IBS patients.Several studies have indicated that enhancing the expression of genes involved in tight junction signaling is a possible mechanism to reinforce intestinal barrier integrity.For instance,lactobacilli modulate the regulation of several genes encoding adherence junction proteins,such as E-cadherin and ?-catenin,in a T84 cell barrier model.Moreover,incubation of intestinal cells with lactobacilli differentially influences the phosphorylation of adherence junction proteins and the abundance of protein kinase C(PKC)isoforms,thereby positively modulating epithelial barrier function.A link between altered levels of pro-inflammatory cytokines and intestinal permeability has been described in a number of intestinal diseases.Using probiotics,the prevention of cytokine-induced epithelial damage,may also contribute to the reinforcement of the mucosal barrier.Further study focused on Lactobacillus rhamnosus GG(LGG)found that LGG prevented cytokine induced apoptosis in intestinal epithelial cells.Follow-up studies resulted in identification of two novel proteins secreted by LGG,termed p40 and p75,that reproduced the effects of live bacteria on cytokine-induced epithelial cell apoptosis and protected the epithelial barrier from peroxide-induced damage.Activation of the PI3K/Akt signaling pathway and PKC-and MAPK-dependent signaling were implicated in these two respective mechanisms of protection.Identification of soluble factors mediating the beneficial effects of probiotics may present an opportunity not only to understand their mechanism of action,but also to develop effective pharmacological strategies that would circumvent many of the problems posed by therapy using live bacteria.It has been suggested that the mechanistic differences between human diseases modeled in animals(mostly rodents)and the pathophysiology of human diseases might be one of the critical factors that contribute to drug failure in clinical trials.Rapid progress in the field of human stem cell technology has allowed the in-vitro recreation of human tissue that should complement and expand upon the limitations of cell and animal models currently used to study human diseases.Recent success in the identification and isolation of human intestinal epithelial stem cells(Lgr5+)from the small intestine has led to culture of functional intestinal epithelial units termed enteroids which composed of crypts but without pericryptal myofibroblasts.Intestinal enteroids are comprised of all four types of normal epithelial cells and develop a crypt–villus differentiation axis.These experimental model systems constitute useful tools for studying the regulation of gastrointestinal stem cells as well as the proliferation and the differentiation of the intestinal epithelial cells throughout the digestive tract.MethodsIn our project,by using an enteroid model system,we showed that the supernatant of Lactobacillus rhamnosus GG(LGG)prevented IFN gamma-induced epithelial barrier damage.The beneficial effects occurred independently of the immune modulating effects of the probiotic.However,the identity of the secreted substance remains unknown.To characterize the secreted protein by LGG we focused on p40 since this is a major band seen on SDS-PAGE analyses of the concentrated LGG supernatant.The coding sequence for P40 was PCR amplified from LGG genomic DNA and subcloned into the pET28b+ expression vector.A 6x histidine-tag was incorporated into the N-terminal of P40.Sequence analysis revealed the presence of a 1236 bp open reading fame,predicted to encode a 412 amino acid residure protein with a calculated MW of 42 kd.The protein was produced using the E.coli strain BL21(DE3)/pLysS and purified by nickel nitrilotrialetic acid agarose and fast protein liquid chromatography.The purified P40 was used to produce specific antibody for subsequent studies.Western blot revealed large quantity of P40 secreted into the supernatant by LGG.We employed human enteroid to investigate whether p40 is responsible for improving mucosal permeability in the absence of immune modulation.ResultsINF gamma was used to induce epithelial barrier damage by causing disruption of tight junction.Incubation with INF gammafor 24 h caused a 40% and 30% downregulation of gene expression of occludin and ZO-1.These changes were prevented by P40.Immunodepletion of P40 from LGG supernatant abolished the abilities of the supernatant to prevent downregulation of ZO-1 and occludin.In separate studies we used in-outside human enteroid structures to evaluate the permeability of the epithelium.Human enteroids were injected with the fluorescence dye FD4 and images were obtained at different time points up to 24 h.Under control conditions,the human enteroids retained 50% of FD4 over 24 h,while treatment of the enteroids with INF gamma impaired permeability resulting in 20% retention of the dye.Administration of P40 prevented leakage of dye evoked by INF gamma.In conclusion,P40 is the first probiotic bacterial product demonstrated to promote junction protein expression and maintain mucosal epithelial barrier function.Our findings provide a molecular basis for therapeutic application of probiotic bacterial products on inflammation-mediated intestinal disorders.Conclusion :1.Generation and purification of p40 from LGG supernatant.2.LGG supernatant only enhances tight junction protein expression and prevents IFN gamma-induced epithelial barrier damage in human enteroids.3.p40 enhances tight junction protein expression and prevents IFN gamma-induced epithelial barrier damage in enteroids.4.Immunodepletion of p40 From LGG-s Blocks LGG-s biologic effects on enteroids.Immunodepletion of P40 fail to increase ZO-1 and occludin mRNA expression in human enteroids.Immunodepletion of p40 from LGG supernatant fail to improve epithelial function impaired by IFNγ in huamn enteroids5.p40 on barrier integrity and epithelial barrier function is in the absence of immune modulation. |
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