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Inflammatory bowel disease (IBD), as an idiopathic inflammatory disease of the intestinal tract, consisting mainly of Crohns disease and ulcerative colitis, which can involve the rectum, colon and ileum, and whose pathogenesis is still not fully understood. The initiation of intestinal inflammation associated with IBD and its chronieity begins with increased intestinal permeability caused by intestinal epithelial barrier disruption. The anti-permeability of the intestinal epithelial barrier is maintained by tight junction in the apical region of the intestinal epithelial cells, and disruption of the tight junction structure is closely associated with intestinal epithelial barrier damage and the development of IBD. Therefore, it is significant to find drugs for the prevention and treatment of IBD using tight junctions as regulatory targets. In recent years, many small molecules of natural product origin have been reported to improve the effects of IBD. In particular, we review the compounds that have the function of repairing intestinal epithelial barrier and protecting tight junction structure, in order to provide research ideas for the design and development of new drugs for the prevention and treatment of IBD.
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Due to incomplete function of gastrointestinal barrier, children are more likely to develop gastrointestinal dysfunction.The clinical application of related biomarkers helps early diagnosis and treatment of gastrointestinal dysfunction in children.By sorting out the studies in recent years, we explored the relationship between inflammatory indicators, intestinal epithelial barrier damage biomarkers, immunological biomarkers, gut microbiome and gastrointestinal dysfunction, and summarized the main problems and solutions faced in the research, which may help the screening, identification and clinical application of relevant biomarkers in subsequent research.
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Objective:To investigate the protective role of Yes-associated protein(YAP)in intestinal epithelial barrier injury.Methods:The intestinal epithelial barrier model was established by culturing human colorectal adenocarcinoma cell line Caco-2 cells, which were divided into four groups: control group, Caco-2 monolayers did not receive any treatment; recombinant human tumor necrosis factor-α(rhTNF-α)group, 100 μg/L of rhTNF-α was added to Caco-2 monolayers; vector+ rhTNF-α group, Caco-2 monolayers were first added with control plasmid pcDNA3.1-vector, and 100 μg/L rhTNF-α was added 24 hours later; YAP+ rhTNF-α group, Caco-2 cells with barrier construction were first added with pcDNA3.1-YAP, and 100 μg/L rhTNF-α was added 24 hours later.Realtime-PCR and Western blot were used to evaluate YAP mRNA and protein expression level.Epithelial permeability was assayed by trans-epithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran 40(FD-40 flu). Cellular distribution of F-actin was assayed by immunofluorescence staining.Results:Compared with control group[(607.3±29.3)Ω·cm 2], TEER of rhTNF-α group[(265.3±32.7)Ω·cm 2] decreased, while TEER of YAP+ rhTNF-α group[(387.0±18.7)Ω·cm 2]increased compared with rhTNF-α group, the differences were statistically significant( P<0.001). The FD-40 flux of rhTNF-α(22.7%±0.5%) group was higher than that of the control group(6.3%±0.9%), while the FD-40 flux of Yap + rhTNF-α group(12.2%±0.8%) was lower than that of rhTNF-α group, the differences were statistically significant( P<0.001). Immunofluorescence staining showed that compared with the control group, the cytoskeletal F-actin fiber dense spot decreased in rhTNF-α group, and some cells showed obvious trans-cellular stress fiber structure, while the peripheral actin band was clear in YAP+ rhTNF-α group, and the intracellular stress fiber decreased.YAP+ TNF-α group appeared as a clear, peripheral actin ribbon with a decrease in cytoplasmic stress fibres. Conclusion:YAP overexpression significantly inhibits TNF-α induced decline of TEER, and increases of FD-40 flux and F-actin rearrangement of Caco-2.YAP could ameliorate TNF-α induced intestinal epithelial barrier injury by regulating cytoskeleton F-actin.
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MicroRNAs (miRNAs) are non-coding single-stranded small RNAs composed of 19 ～25 nucleotides involved in various life processes such as cell proliferation,differentiation,apoptosis,signal transduction and immune regulation at the post-transcriptional level.So it has become one of the research hotspots in recent years.Among them,MiR-7 is considered to be an important regulator of epithelial cell damage and repair.As the largest epithelium of the human body,the intestinal macosal epithelium is the link between the human body and the outside world,and plays an important role in the homeostasis of the internal environment.Numerous studies have shown that MiR-7 plays an irreplaceable role in intestinal epithelial barrier damage and repair through the epidermal growth factor receptor (EGFR)-related signal pathway.This paper reviews the research progress.
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MicroRNAs are a group of large and highly conserved non-coding small stranded RNAs that regulate the gene expression by translational inhibition or mediated degradation of target proteins at post-transcriptional levels,which plays an important role in many biological processes,including cell apoptosis,immune response,tumorigenesis and nutritional metabolism.The intestinal epithelial barrier is the first defense to maintain the general physiological phenomenon,and its integrity is a critical factor of the maintenance of intestinal mucosal barrier function and intestinal homeostasis.This article reviews the biological function of miRNAsand their post-transcriptional regulation of the formation of intestinal epithelial barrier.
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Heatstroke impairs the intestinal mucosal barrier function,leading to bacteremia,endotoxemia and multiple organ dysfunction.Therefore,protecting intestinal barrier function is critical in the prevention and treatment of heatstroke.Gut microbiota has an important impact on intestinal mechanical,biological and immune barrier.Intestinal dysbacteriosis damages intestinal b,arrier,which leads to enterogenous bacteria and endotoxin from the intestinal lumen entering into the circulation and thus causes systemic inflammatory response syndrome.This process can trigger heatstroke and promote its development and progression.However,probiotics can reduce endotoximia and inflammatory cytokines by improving the intestinal barrier function.Therefore,according to the effect of gut microbiota on the relevant aspects of intestinal mucosal barrier,searching for target probiotics and intervention may be an effective strategy for the prevention and treatment of heatstroke against intestinal barrier damage.
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Aim To investigate the protective effect of paeoniflorin on TNF-α induced intestinal epithelial barrier dysfunction and its mechanisms.Methods The Caco-2 cells were cultured and the MTF assay was used to determine the effects of the paeoniflorin on Caco-2 cell activity.The Caco-2 cell intestinal epithelial barrier dysfunciton model was established through incubation of cells with TNF-α.The effects of paeoniflorin on intestinal epithelial barrier dysfunciton were studied.Results The transmembrane resistance in Caco-2 epithelial barrier was significantly reduced by TNF-α incubation;MLCK significantly increased,while tight junction protein occludin and ZO-1 significantly decreased by TNF-α.These changes were significantly reversed by paeoniflorin,which reduced MLCK expression and enhanced expression of occludin and ZO-1.The protective effects against epithelial barrier dysfunction could be abrogated by small interfering RNA(siRNA) of MLCK.Conclusions Paeoniflorin alleviates the epithelial barrier dysfunction induced by TNF-αthrough down-regulation of MLCK and enhancement of tight junction protein occludin and ZO-1.This study supplies a potential candidate drug for the clinical treatment of inflammatory bowel diseases.
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Objective Intestinal epithelial barrier damage is closely related to a variety of gastrointestinal disease,how to maintain its function effectively is the key to treat all these diseases.This research attempts to explore the protective effect and its mechanism of toll-like receptor 2 (Toll-like receptor,TLR2)on permeability of intestinal epithelial barrier by experiments in vitro,to lay a foundation for new treatment methods.Methods We cultured non-transfected Caco-2 cells,TLR2-deficiency Caco-2 cells,TLR2-overexpressed Caco-2 cells in normal control group until the 21 st d,then tested transepithelial electrical resistance(TEER) which reacts the permeability of epithelial barrier.We cultured 3 types of cells in inflammation group until the 19th d treated with 10 ng/ml IL-1 beta for 48 h,then tested TEER values at the 21st d.We treated 3 types of cells in inhibition group with PI3K/Akt pathway inhibitor for 1h befor IL-1 beta,then tested TEER values at the 21st d.Results TEER value of TLR2-deficiency Caco-2 cell monolayer significantly reduced (P ＜ 0.01),whereas TEER value of TLR2-overexpressed Caco-2 monolayer raised,but without statistically significant.TLR2 can prevent IL-1 beta caused TEER decreasing (P ＜ 0.01),but the effect disappeared after given PI3K/Akt pathway inhibitor.Conclusion TLR2 can regulate the permeability of intestinal epithelial barrier.In addition,TLR2 can protect permeability increasing caused by inflammation,this effect mediated by PI3 K/Akt pathway.
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Objective To investigate the effect of adrenomedullin on epithelial barrier function of Caco-2 cells under hypoxia/reoxygen-ation injury and the molecular mechanism.Methods Build hypoxia/reoxygenation injury model,Caco-2 cells were randomly divided into three groups:normal control group( N) ,hypoxia/reoxygenation group( H/R) and hypoxia/reoxygenation plus 1 μmol/L adrenomedullin group(H/R+AM).Transepithelial electrical resistance(TEER) was determined,and the laser confocal microscope was used to detect the construction of tight junction.The protein expressions of tight junction proteins (Occludin and ZO-1) and nuclear factor kappa B(NF-κBp65) were examined by using western blotting.Results Pretreatment of AM significantly attenuated the disruption of morphological structure of tight junction caused by H/R.The TEER of N group,H/R group and H/R+AM group were(157.68 ±5.54),(96.06 ±3.42),(134.56 ± 4.72) Ohm/cm2 respectively.After AM pretreatment, the TEER were significantly raised compared with that in H/R group by 40.00%(P<0.05).The relative protein expressions of Occludin and ZO-1 in N group,H/R group and H/R+AM group were [(0.43 ±0.03), (0.27 ±0.04)],[(0.20 ±0.03),(0.15 ±0.07)],[(0.32 ±0.15),(0.21 ±0.03)]respectively.After AM pretreatment,the protein ex-pression of Occludin and ZO-1 were significantly raised compared with that in H/R group(by 60.00% and 40.00%).Simultaneously,the relative protein expressions of NF-κBp65 in N group,H/R group and H/R+AM group were (0.53 ±0.30),(2.89 ±0.16),(1.75 ±0.25) respectively.And H/R induced NF-κBp65 activation was significantly inhibited by AM treatment (decreased as compared with H/R group by 40.00%).Conclusion AM can attenuate intestinal epithelial barrier dysfunction by regulating the expression of intestinal tight junction pro-teins.The inhibition of NF-κB activation may be involved in this mechanism.
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Objective To investigate the role of Rho kinase (ROCK) in the protective effects of hydrogen on intestinal epithelial barrier function in sepsis. Methods Caco-2 cells were cultured routinely, and divided into 6 groups randomly (n=3):control group (C group), hydrogen-rich medium group (H group), lipopolysaccharide (LPS)-treatment group (L group), hy?drogen+LPS-treatment group (HL group), Rho kinase inhibitor (Y-37632) treatment group (Y group) and Rho kinase inhibi?tor Y-27632+LPS-treatment group (YL group). H group was treated with 0.6 mmol/L hydrogen-rich media. The concentra?tion of LPS and Y-27632 were 50 mg/L and 25μmol/L separately. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured regularly. When the TEER value reached 800Ω·cm2, the treatment was administered. Then TEER values were measured at 6 h, 12 h and 24 h, and FITC-dextran permeability was de?tected at 24 h. Cells were seeded on 6-well plates. After cell density reached 80%-90%, treatments were given randomly. The real time-polymerase chain reaction (RT-PCR) was conducted to assess mRNA levels of ZO-1 and ROCK mRNA. ZO-1 and ROCK protein expression levels were detected by Western blot assay. Results Compared with C group, TEER values were elevated in 12 h and 24 h in H group (P protein expression levels of ZO-1 and ROCK between C group and H group (P>0.05). TEER values were elevated at 6 h, 12 h and 24 h in Y group (P 0.05). The mRNA expression of ZO-1 increased and mRNA expression of ROCK decreased in Y group (P <0.05). The TEER values reduced at 6 h, 12 h and 24 h in L group. The FITC-dextran permeability increased significantly, mRNA and protein expressions of ZO-1 significantly decreased, mRNA and protein expressions of ROCK significantly in?creased in L group (all P<0.05). Compared with L group, TEER values increased significantly at 6 h, 12 h and 24 h in YL group, FITC-dextran permeability decreased, mRNA expressions of ZO-1 increased, mRNA expressions of ROCK de?creased in YL group (P<0.05). Compared with L group, TEER values increased at 6 h, 12 h and 24 h in HL group, FITC-dextran permeability reduced markedly, protein expressions of ZO-1 increased at each time point, protein expressions of ROCK decreased at each time point in HL group (P<0.05). Conclusion Hydrogen can protect intestinal barrier function against sepsis, ameliorate the integrity and permeability of intestinal epithelium and increase the expressions of intercellular tight junction proteins. The suppression of Rho kinase over-expression induced by LPS may be involved in these protective effects of hydrogen.
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AIM:To investigate the effect of microRNA-429 (miR-429) on the expression of occludin (Ocln) in intestinal epithelial cells ( IECs) and intestinal epithelial barrier function in diabetic mice.METHODS:Diabetes mel-litus mouse model was induced by intraperitoneal injection of streptozocin.The expression of miR-429 in IECs of diabetic mice was inhibited by antagomiRNA-429 injected via tail vein.The expression of miR-429 and mRNA expression of Ocln were detected by real-time PCR.The protein expression of Ocln was determined by Western blotting and immunohistochem-istry.The urinary lactulose/mannitol ratio was measured by gas chromatography.The plasma LPS concentrations in the mice were measured by chromogenic end-point TAL kit.RESULTS:The results of real-time PCR confirmed that the ex-pression of miR-429 in IECs of diabetic mice was remarkably inhibited by tail-vein administration of antagomiRNA-429, and resumed to similar level of normal mice on the 6th day after the administration.After suppressing the level of miR-429, the expression of Ocln in IECs of diabetic mice increased significantly (P<0.05), while the urinary lactulose/mannitol ra-tio and the plasma LPS concentration decreased obviously ( P<0.05 ) .CONCLUSION:AntagomiRNA-429 effectively suppresses miR-429 expression in IECs of diabetic mice, and then enhances the expression of Ocln and partially resumes the intestinal epithelial barrier function.
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Objective To investigate the effect of S-Nitrosoglutathione (GSNO)on acute ischemia reperfusion (I/R)induced in-testinal barrier function lesion in a mouse model.Methods Twenty-four 6-8-year-old C57BL/6 mice were divided into 3 groups,8 for each:(1)the sham group;(2)the I/R group;(3)the I/R+GSNO group.The mouse intestine I/R model was established by the occlusion of the superior mesenteric artery temporarily followed by reperfusion for 6 h.Histological changes in the small intestine were observed after HE staining;the expression of claudin-1 protein in the intestine epithelium was assessed by immunohistochem-istry staining as well as western blot analysis.Results Both HE staining and immunohistochemistry results showed the integrate intestinal villi with the continuous Claudin-1 expression alone the villi in the sham group;the intestinal villi of the I/R group partial-ly detached,thickened,crooked and fractured,with the obvious disconnection of Claudin-1 staining alone the top of the villi;while the intestinal villi of the I/R+GSNO group were neatly arranged and damage to intestinal mucosa was much alleviated,accompanied with the marked restoration of the continuity of claudin-1 staining.Compared to the sham group,claudin-1 protein level of for the I/R group and the I/R+GSNO group decreased by 32.5% and 13.8% respectively (P <0.05);and compared to the I/R group,clau-din-1 protein level of the I/R+GSNO group increased by 27.8% (P <0.05).Conclusion Protein level of claudin-1 would decrea-ses after I/R,and pretreatment with GSNO can effectively relieve the damage of intestinal mucosal structure as well as intestinal tight junction barrier through upregulating the expression of claudin-1 protein.
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Objective A stable rat model of heat stroke was established to investigate heat stroke-induced changes of the intestinal epithelial tight junction (TJ) barrier permeability, and to investigate the mechanism by observing the changes of TJ protein (occludin) expression and TJ morphology. Methods SD rats were randomly divided into two groups(n = 10): heat stroke group and normal control group. Stable model of heat stroke was established. Anesthetized rats were exposed to 42 ℃ in a ventilated chamber for 50min, after heat exposure, the rats were placed in room temperature(26 ℃ ) for 2 h. Then the rats were sacrificed and samples were taken. The effect of heat stroke on intestinal epithelial barrier permeability was observed through changes of plasma FD4 and endotoxin concentration, cytokines concentration was detected as inflammatory indicators. The general and micro pathology was observed by light microscope (hematoxylin and eosin-staining, HE staining) and transmission electron microscope (TEM). Occludin expression was investigated by Western bloting and immunochemistry. Results At 2 h after heat exposure, intestinal epithelial barrier permeability( FD4 and endotoxin concentration), and cytokines levels of heat stroke group were significantly higher than normal control group(P ＜ 0. 05 ). In morphology, by observing the light micrographs of HE jejunal tissue, the sloughing of epithelium off the basement membrane at the villus tips of the heat stroke group compared with the normal controlgroup. In many visual fields (VF)( ≥6VF/slice), this phenomenon was not universal. Under TEM, TJ of normal enterocytes was integrated with the compact zonal structure. At 2 h after heat exposure, TJ of heat stroke group was broken with widen intercellular space, and the density of TJ was decreased. In immunochemistry assay, the positive signal was distributed along the cell membrane in normal small intestinal tissue, while the signal was obviously decreased in heat stroke group.The results of Western bloting showed that occludin expresion of heat stroke group was significantly lower than normal control group(P ＜0.05). Conclusion Heat stroke decreased occludin expression, opened the intestinal epithelial TJ, which destroyed intestinal epithelial tight junction barrier, induced high intestinal epithelial barrier permeability, produced endotoxemia and systemic inflammatory response syndrome (SIRS).
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The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-á) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-á (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-á treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-á decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-á did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-á increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.
Subject(s)
Humans , Cell Membrane Permeability/drug effects , Epithelial Cells/drug effects , Intestinal Mucosa/cytology , Membrane Proteins/drug effects , Tight Junctions/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Blotting, Western , Epithelial Cells/metabolism , Membrane Proteins/metabolism , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tight Junctions/metabolismABSTRACT
After birth,human intestinal tract mucosa is exposed to a large community of commensal and pathogenic bacteria. As a first line of defense,the intestinal epithelial barrier (IEB) kills the pathogen by signaling to the innate immune system, through pattern recognition receptors, while it produces protective respond towards the commensal bacteria. Intestinal epithelial cells play an important role in forming immune tolerance to the commensal bacteria and make intestinal homeostasis. Commensal bacteria can resist the pathogenic bacteria invasion. The signals of commensal are required for development of intestinal epithelial barrier and intestinal innate and adaptive immunity. It is essential for the host to have a balance between the commensal bacteria and intestinal tract,once the balance is broken, the intestinal inflammation disease will be caused. Thus ,this review will discuss the relationship between intestinal commensal bacteria and intestinal epithelial barrier in several aspects, such as the role of the commensal bacteria, the mechanism of producing commensal tolerance by IEB and the disease caused by imbalance between the commensal and IEB.