Intestinal Epithelial Barrier Dysfunction Research Articles

Heat-killed yogurt-containing lactic acid bacteria prevent cytokine-induced barrier disruption in human intestinal Caco-2 cells

Probiotics have been proven to play a protective role against gut barrier disruption induced by enteroinvasive pathogens and chemical substances. To test the hypothesis that a heat-inactivated multi-strain of yogurt-containing lactic acid bacteria (Mix-LAB) (Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus) could prevent epithelial barrier dysfunction induced by inflammatory cytokines, human intestinal Caco-2 cell monolayers were cultured for 72 h with cytomix (interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta) alone and in combination with graded concentrations of heat-inactivated Mix-LAB (107,108,109/ml). After incubation for 72 h, cytomix decreased the transepithelial electrical resistance (TEER) and increased the paracellular permeability of Caco-2 monolayers to fluoresceinated dextran (molecular weight 376 Da), which was inhibited by the pretreatment of heat-inactivated Mix-LAB under culture conditions. This protective effect of heat-inactivated Mix-LAB on reduced TEER and increased paracellular permeability was concentration-dependent, rising to statistically significant levels for concentrations equal to or greater than 107 /ml after 72-h incubation and 108 /ml after 48-h incubation. Cytomix altered the expression and distribution of the tight junction proteins ZO-1 and occludin, but this effect was prevented by the heat-inactivated Mix-LAB. These data suggest that heat-inactivated yogurt-containing Mix-LAB plays a protective role in proinflammatory cytokine-induced intestinal epithelial barrier dysfunction.

Mitogen-activated protein kinase phosphatase-1 prevents lipopolysaccharide-induced apoptosis in immature rat intestinal epithelial cells.

Necrotizing enterocolitis is characterized by intestinal inflammation and epithelial barrier dysfunction. Mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 plays a pivotal role in the feedback control of MAPK signaling, which regulates inflammation and apoptosis. We hypothesized that MKP-1 prevents lipopolysaccharide (LPS)-induced apoptosis in intestinal epithelial cells. Western blot analysis and qPCR were used to assess MKP-1, MAPK (p38, extracellular signal-regulated kinase (ERK), and c-Jun N terminal kinases (JNK)), caspase 3, caspase 9, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 expression levels in rIEC-6 enterocytes. MKP-1 expression was inhibited using small interfering RNA (siRNA) methodology. Viable cell number was determined using trypan blue exclusion. LPS stimulation led to activation of p38, JNK, and ERK, and induction of MKP-1 mRNA and protein expression. The induction of MKP-1 was associated with a decrease in p38 phosphorylation, and knockdown of MKP-1 prolonged p38 phosphorylation. While LPS stimulation significantly attenuated proliferation of rIEC-6 cells transfected with scramble siRNA, LPS stimulation resulted in a net decrease in viable cell number in cells transfected with MKP-1 siRNA. Following LPS stimulation, MKP-1 knockdown resulted in greater caspase 3 and 9 activities and greater proinflammatory cytokine (TNF-α, COX-2) expression than in cells transfected with scramble siRNA. Our results demonstrate that MKP-1 has a central role in preventing inflammation-induced apoptosis in rIEC-6 enterocytes.

Tu1374 1,25-Dihydroxyvitamin D3 Preserves Intestinal Epithelial Barrier Function From TNF-α Induced Injury via Suppression of MLCK-Mlc Phosphorylation Signaling Pathway

Intestinal barrier dysfunction occurs inmultiple enteropathies. Some known proinflammatory cytokines, such as TNF-a, INF-γ, play a pivotal role as mediator between various pathogens and gut epithelium. In our previous work, TNF-α injured Caco-2 monolayers were used to screen potential barrier protective reagents. 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) turned out to be a very effective protector. This is consistent with literatures which report that 1,25(OH)2D3 has anti-inflammation effect in bowel diseases. To elucidate the underlying mechanism, we performed this work. Given the fact that tight junction (TJ) is the important intercellular structure related with intestinal epithelial barrier function, and that the key role of myosin light chain (MLC) phosphorylation in regulating TJ. The TJ status and MLC phosphorylation was studied.Methods Experiments were conducted on Caco-2 monolayers. Barrier dysfunction was generated by 10ng/ml TNF-α treatment for up to 48h. Barrier function was represented by trans-epithelial electrical resistance (TEER), and macro molecule tracer, FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. Morphological changes of intercellular TJ were visualized by immunofluorescence staining of ZO-1 and Occludin. The expression level of phosphorylated MLC (P-MLC), and its upstream MLC kinase (MLCK) were determined by immunoblotting. The nuclear translocation and activation of NF-kB p65 was analyzed by Electrophoretic Mobility Shift Assay (EMSA) and immunofluorescent staining. Results TNF-α decreased TEER by ~60% after 48h treatment, 10-7M 1,25(OH)2D3 reversed TEER to ~80% of original level. Consistently, TNF-α increased FD-40 flux through monolay by nearly 5 folds, 1,25(OH)2D3 repressed the number to about2 folds. TJ structure altered significantly following TNF-α treatment. 1,25(OH)2D3 ameliorated TNF-α-induced TJ damage. The MLC phosphorylation, as well as MLCK protein expression were induced by TNF-α. These increases were significantly inhibited by 1,25(OH)2D3 treatment. Additionally, 1,25(OH)2D3 suppressed the nuclear translocation and activation of NF-kB p65.Conclusion 1,25(OH)2D3 attenuates TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway ofMLCK-dependentMLCphosphorylation via NF-kB p65 pathway.

Loss of Cortactin is Associated with Intestinal Epithelial Barrier Dysfunction and Development of Colitis

A stable intestinal epithelium is the basis for healthy intestines. Its dysfunction causes increased intestinal permeability and bacterial translocation that may eventually cause an aberrant immune response leading to chronic inflammatory diseases of the intestines such as inflammatory bowel disease (IBD). The actin cytoskeleton regulates epithelial barrier integrity but the role of actin regulators such as cortactin (CTTN) is not well understood. Analyzing CTTN‐KO mice, we found increased basal intestinal permeability similar to what has been shown in the vasculature. Using a CTTN‐depleted Caco‐2 cell line, we confirmed epithelial dysfunction by reduced transepithelial resistance (TER), a weaker cortical actin ring, internalization of occludin and ZO‐1 and accelerated transmigration of neutrophils. Calcium‐switch assays revealed a strong delay in barrier formation without CTTN. While CTTN depletion did not cause increased apoptosis, we observed increased proliferation. Importantly, CTTN localized at cell contacts in colon biopsies of healthy individuals, whereas in IBD patients we observed strong translocation of CTTN into the cytosol and loss of colocalization with actin and ZO‐1. Our data imply that CTTN controls intestinal barrier integrity under basal conditions and that loss of CTTN triggers epithelial dysfunction as seen in IBD.

The Pregnane X Receptor Attenuates Cytokine‐Induced Intestinal Epithelial Barrier Dysfunction, an Effect Associated With Inhibition of JNK Activation and Enhanced Autophagy

The Pregnane X Receptor Attenuates Cytokine‐Induced Intestinal Epithelial Barrier Dysfunction, an Effect Associated With Inhibition of JNK Activation and Enhanced Autophagy

Tu1852 Resveratrol Ameliorates Oxidative Stress-Induced Intestinal Epithelial Barrier Dysfunction by Upregulating Heme Oxygenase-1 Expression

Crohn's disease (CD) is a chronic, relapsing inflammatory disease, which affects nearly 1.4 million Americans. Genome-wide association studies (GWAS) have identified several genetic risk loci for CD including IRGM (immunity-related GTPase family M). IRGM functions in the innate immune control of intracellular pathogens. IRGM localizes to the mitochondria and induces mitochondrial fission which is linked to the autophagic elimination of intracellular bacteria. Surprisingly, IRGM expression is not lost but stronger in the epithelium of CD patients carrying the IRGM risk allele and more unexpectedly it is associated with an increase in intracellular bacteria. So how IRGM signaling contributes to the pathogenesis of CD remains unresolved. Our laboratory has generated a unique mouse model (DKO) of CD with a high mucosal expression of IRGM which duplicates the functional, histological and clinical features of human CD. The DKO mice were generated by genetic deletion of two anti-apoptotic proteins (villin/gelsolin) that regulate mitochondrial homeostasis and mucosal healing. Our studies with DKO mice and human CD patients demonstrate that by acting on the mitochondria, IRGM confers autophagic protection when expressed in moderation but induces cell death when over-expressed, explaining IRGM's action both in defense against bacteria and in damaging inflammation in CD. Furthermore, IRGM induced cell death is accompanied by the release of HMGB1 (high mobility group protein B1), a major proinflammatory alarmin/DAMP (danger-associated molecular pattern). This we demonstrate is the molecular basis of IRGM's role in the pathogenesis of CD. Our innovative study uses a novel mouse model of CD, provides a novel paradigm about the role of IRGM in CD and includes translational studies to diagnose, treat and monitor CD patients.

Par-3 modulates intestinal epithelial barrier function through regulating intracellular trafficking of occludin and myosin light chain phosphorylation.

Tight junctions play a critical role in the maintenance of intestinal barrier function. Partitioning-defective protein 3 (Par-3) can regulate intestinal barrier function through the modulation of tight junction assembly and cell polarity. However, the mechanisms are still not fully understood. Adult C57BL/6 mice were treated with dextran sulfate sodium for 7 days, and segments of colon were harvested for immunofluorescent staining of Par-3. Caco-2 intestinal epithelial cells were treated with tumor necrosis factor α (TNF-α) for 24 h, and Par-3 expression was detected by Western blot analysis and immunofluorescence. Additionally, Caco-2 cells were treated with Par-3 small interfering RNA, and altered expression and subcellular localization of tight junction proteins were studied by Western blot analysis and immunofluorescence. Furthermore, the interaction between Par-3 and myosin light chain (MLC) was detected by immunoprecipitation. Par-3 was downregulated in murine dextran sulfate sodium induced acute inflammation and TNF-α-treated Caco-2 cells. Depletion of Par-3 expression by small interfering RNA delayed intestinal epithelial barrier development in Caco-2 cells. This regulation was due to the redistribution of the tight junction protein occludin rather than the altered total levels of tight junction proteins. Par-3 silencing blocked the trafficking of occludin from or through the Golgi complex to the cell surface, and dramatically induced occludin accumulated at the Golgi complex. Importantly, Par-3 can interact with MLC, and loss of Par-3 upregulated MLC kinase expression and MLC phosphorylation, which contributed to intestinal epithelial barrier dysfunction. These results indicate that Par-3 plays an important role in the modulation of intestinal barrier function by regulating delivery of occludin as well as suppression of MLC phosphorylation.

Oregano essential oil decreased susceptibility to oxidative stress-induced dysfunction of intestinal epithelial barrier in rats

The protective effects of oregano essential oils (OEO) against oxidative stress in intestine of rats were investigated. The results showed that rats orally treated with 5 or 20 mg/kg BW with OEO abolished diquat-induced oxidative stress in jejunum, thereby maintaining jejunal architecture. The protective effect of OEO might cause modification of selected intestinal microbiota, inhibition of inflammatory cytokine expression and subsequent increased occludin expression in jejunum by enhancement of antioxidative capacity. These results revealed that OEO exerted a protective effect against diquat-induced oxidative injury in intestine of rats.

Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

Clinical characteristics associated with postoperative intestinal epithelial barrier dysfunction in children with congenital heart disease.

Children with congenital heart disease have loss of intestinal epithelial barrier function, which increases their risk for postoperative sepsis and organ dysfunction. We do not understand how postoperative cardiopulmonary support or the inflammatory response to cardiopulmonary bypass might alter intestinal epithelial barrier function. We examined variation in a panel of plasma biomarkers to reflect intestinal epithelial barrier function (cellular and paracellular) after cardiopulmonary bypass and in response to routine ICU care. Prospective cohort. University medical center cardiac ICU. Twenty children aged between newborn and 18 years undergoing repair or palliation of congenital heart disease with cardiopulmonary bypass. We measured baseline and repeated plasma intestinal fatty acid-binding protein, citrulline, claudin 3, and dual sugar permeability testing to reflect intestinal epithelial integrity, epithelial function, paracellular integrity, and paracellular function, respectively. We measured baseline and repeated plasma proinflammatory (interleukin-6, tumor necrosis factor-α, and interferon-γ) and anti-inflammatory (interleukin-4 and interleukin-10) cytokines, known to modulate intestinal epithelial barrier function in murine models of cardiopulmonary bypass. All patients had abnormal baseline intestinal fatty acid-binding protein concentrations (mean, 3,815.5 pg/mL; normal, 41-336 pg/mL). Cytokine response to cardiopulmonary bypass was associated with early, but not late, changes in plasma concentrations of intestinal fatty acid-binding protein 2 and citrulline. Variation in biomarker concentrations over time was associated with aspects of ICU care indicating greater severity of illness: claudin 3, intestinal fatty acid-binding protein 2, and dual sugar permeability test ratio were associated with symptoms of feeding intolerance (p < 0.05), whereas intestinal fatty acid-binding protein was positively associated with vasoactive-inotrope score (p = 0.04). Citrulline was associated with larger arteriovenous oxygen saturation difference (p = 0.04) and had a complex relationship with vasoactive-inotrope score. Children undergoing cardiopulmonary bypass for repair or palliation of congenital heart disease are at risk for intestinal injury and often present with evidence for loss of intestinal epithelial integrity preoperatively. Greater severity of illness requiring increased cardiopulmonary support rather than the inflammatory response to cardiopulmonary bypass seems to mediate late postoperative intestinal epithelial barrier function.

A Novel Role of OS-9 in the Maintenance of Intestinal Barrier Function from Hypoxia-induced Injury via p38-dependent Pathway.

OS-9 is a lectin required for efficient ubquitination of glycosylated substrates of endoplasmic reticulum-associated degradation (ERAD). OS-9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. However, we know very little about other functions of OS-9 under endoplasmic reticulum stress. Here, we used gene knockdown and overexpression approaches to study the protective effect of OS-9 on intestinal barrier function of intestinal epithelial cell Caco-2 monolayer. We found that OS-9 attenuated intestinal epithelial barrier dysfunction under hypoxia through up-regulating occludin and claudin-1 protein expression. Furthermore, we showed that the up-regulation of occludin and claudin-1 induced by OS-9 was mediated by p38 and ERK1/2 phosphorylation and did not involve HIF-1α. In summary, our results demonstrate that OS-9 up-regulates occludin and claudin-1 by activating the MAP kinase (MAPK) pathway, and thus protects the epithelial barrier function of Caco-2 monolayer under hypoxia condition.

Amelioration of hypoxia and LPS-induced intestinal epithelial barrier dysfunction by emodin through the suppression of the NF-κB and HIF-1α signaling pathways.

Intestinal barrier dysfunction occurs in critical illnesses and involves the inflammatory and hypoxic injury of intestinal epithelial cells. Researchers are still defining the underlying mechanisms and evaluating therapeutic strategies for restoring intestinal barrier function. The anti-inflammatory drug, emodin, has been shown to exert a protective effect on intestinal barrier function; however, its mechanisms of action remain unknown. In this study, we investigated the protective effects of emodin on intestinal barrier function and the underlying mechanisms in intestinal epithelial cells challenged with lipopolysaccharide (LPS) and hypoxia/reoxygenation (HR). To induce barrier dysfunction, Caco-2 monolayers were subjected to HR with or without LPS treatment. Transepithelial electrical resistance and paracellular permeability were measured to evaluate barrier function. The expression of the tight junction (TJ) proteins, zonula occludens (ZO)-1, occludin, and claudin-1, as well as that of hypoxia-inducible factor (HIF)-1α, phosphor-IκB-α, phosphor-nuclear factor (NF)-κB p65 and cyclooxygenase (COX)-2 was determined by western blot analysis. The results revealed that emodin markedly attenuated the decrease in transepithelial electrical resistance and the increase in paracellular permeability in the Caco-2 monolayers treated with LPS and subjected to HR. Emodin also markedly alleviated the damage caused by LPS and HR (manifested by a decrease in the expression of the TJ protein, ZO-1), and inhibited the expression of HIF-1α, IκB-α, NF-κB and COX-2 in a dose-dependent manner. In conclusion, our data suggest that emodin attenuates LPS- and HR-induced intestinal epithelial barrier dysfunction by inhibiting the HIF-1α and NF-κB signaling pathways and preventing the damage caused to the TJ barrier (shown by the decrease in the expression of ZO-1).

Homocysteine-mediated intestinal epithelial barrier dysfunction in the rat model of irritable bowel syndrome caused by maternal separation.

Homocysteine-mediated intestinal epithelial barrier dysfunction in the rat model of irritable bowel syndrome caused by maternal separation.

Oxymatrine improves intestinal epithelial barrier function involving NF-κB-mediated signaling pathway in CCl4-induced cirrhotic rats.

Accumulating evidence suggests that intestinal epithelial barrier dysfunction plays an important role in the pathogenesis of hepatic cirrhosis and its complications such as gastrointestinal injury and hepatic encephalopathy. To date, there is no cure for cirrhosis-associated intestinal mucosal lesion and ulcer. This study aimed to investigate the effect of oxymatrine on intestinal epithelial barrier function and the underlying mechanism in carbon tetrachloride (CCl4)-induced cirrhotic rats. Thirty CCl4-induced cirrhotic rats were randomly divided into treatment group, which received oxymatrine treatment (63 mg/kg), and non-treatment group, which received the same dose of 5% glucose solution (vehicle). The blank group (n = 10 healthy rats) received no treatment. Terminal ileal samples were collected for histopathological examination. The expression level of nuclear factor-κB (NF-κB) p65 in ileal tissue was evaluated by immunohistochemistry. The gene and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in ileal tissues were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Additionally, plasma endotoxin level was determined. In comparison to the blank group, a significant alteration in the morphology of intestinal mucosal villi in the non-treatment group was observed. The intestinal mucosal villi were atrophic, shorter, and fractured, and inflammatory cells were infiltrated into the lamina propria and muscular layer. Besides, serious swell of villi and loose structure of mucous membrane were observed. Oxymatrine reversed the CCl4-induced histological changes and restored intestinal barrier integrity. Moreover, oxymatrine reduced the protein expression level of NF-κB p65, TNF-α, and IL-6, which were elevated in the vehicle-treated group. In addition, the serum endotoxin level was significantly decreased after oxymatrine treatment in CCl4-induced cirrhotic rats. The results indicate that oxymatrine improves intestinal barrier function via NF-κB-mediated signaling pathway and may be used as a new protecting agent for cirrhosis-associated intestinal mucosal damage.

GDNF is involved in the barrier-inducing effect of enteric glial cells on intestinal epithelial cells under acute ischemia reperfusion stimulation.

Acute intestinal ischemia reperfusion (IR) injury is often associated with intestinal epithelial barrier (IEB) dysfunction. Enteric glial cells (EGCs) play an essential role in maintaining the integrity of IEB functions. However, the precise mechanism of EGCs under IR stimulation remains unclear. Here, we report that EGCs are closely involved in the modulation of IEB functions in response to IR challenge. The intestinal IR treatment led to the significant upregulation of the EGC activation marker, glial fibrillary acidic protein, accompanied by the increasing abundance of glial-derived neurotrophic factor (GDNF) and inducible nitric oxidase (iNOS) proteins, which was also confirmed in in vitro hypoxia reoxygenation (HR) tests. Co-culturing with EGCs attenuated the tight junctional abnormalities, blocked the downregulation of ZO-1 and occludin protein expression, and relieved the decrease of permeability of intestinal epithelial cell (IEC) monolayers under HR treatment. Furthermore, exogenous GDNF administration displays the barrier-protective effects similar to EGCs against HR stimulation, while RNA interference-mediated knockdown of GDNF significantly inhibited the protective capability of EGCs. The expression of both GDNF and iNOS proteins of EGCs was significantly upregulated by co-culturing with IECs, which was further increased by HR treatment. Interestingly, through inhibiting iNOS activity, the barrier-protective effect of EGCs was influenced in normal condition but enhanced in HR condition. These results suggest that GDNF plays an important role in the barrier-protective mechanism of activated EGCs under IR stimulation, whereas EGCs (via iNOS release) are also involved in intestinal inflammation response, which may contribute to IEB damage induced by IR injury.

Intestinal epithelial barrier dysfunction protects against pathogen translocation during initial infection (1111.2)

Myosin light chain kinase (MLCK) is essential for pathophysiological epithelial tight junction regulation. Transgenic mice with intestinal epithelial‐specific expression of constitutively‐active MLCK (CA‐MLCK) demonstrate increased permeability and susceptibility to immune‐mediated colitis. However, the effect of barrier dysfunction on enteric infection remains unknown. Within 30 min after mucosal application of DsRed‐labeled Salmonella typhimurium (SL3201 108 CFU), CA‐MLCK mice exhibited a 5‐fold reduction in bacterial translocation compared to wildtype (WT). This protection also occurred 1 h after oral infection with Toxoplasma gondii, in which tachyzoite invasion was reduced in CA‐MLCK mice relative to WT, 1.5±0.6 vs 4.0±1.0 tachyzoite/mm2 villus, respectively. Intraepithelial lymphocytes expressing the γδ T‐cell receptor (γδ IEL) protect against pathogen invasion. Time‐lapse confocal microscopy revealed enhanced γδ IEL surveillance of the villous epithelium in CA‐MLCK mice compared to WT with more rapid migration (4.2±0.1 vs 3.8±0.1μm/min) and reduced localization to the lateral intercellular space (26±1.7 vs 32±3.3%). However, loss of γδ T cells in CA‐MLCK mice did not alter Salmonella transmigration compared to WT. These data indicate that increased γδ IEL epithelial surveillance is not sufficient for the reduced pathogen invasion observed during early infection in CA‐MLCK mice.Grant Funding Source: Supported by NIH DK61931 and DK68271, UChicago DDRCC, Gastro‐intestinal Research Foundation

Increased Gap Density Predicts Weakness of the Epithelial Barrier In Vivo by Confocal Laser Endomicroscopy in Indomethacin-Induced Enteropathy

The intestinal epithelial barrier plays an important role in the pathogenesis of non-steroidal anti-inflammatory drug-induced enteropathy, and its disruption is often associated with increased cell shedding. The purpose of this report is to observe the gap density in indomethacin-induced small intestinal damage by confocal laser endomicroscopy (CLE) and to investigate the mechanisms involved in this process and how mucosal protectants improve intestinal epithelial barrier dysfunction. CLE is expected to provide a new way for evaluating non-steroidal anti-inflammatory drugs-induced enteropathy in humans and assessing drug efficacy. Using the new technique of CLE, we established a method to evaluate, in real time, intestinal damage after the administration of indomethacin in Wistar rats by investigating the gap density in the small intestine. The mucosal protectant teprenone and acid-suppressant rabeprazole were then given by gavage before and after the administration of indomethacin, and the mechanisms affecting the intestinal epithelial barrier were investigated. Using CLE, gaps could be clearly observed and easily distinguished from goblet cells. Gap density was increased after the administration of indomethacin. During this process, the expression of tumor necrosis factor-α, nuclear factor-κB, and caspase-3 was up-regulated and the expression of tight junctions was down-regulated, which led to the damage of the epithelial barrier. Teprenone and rabeprazole could intervene in this pathway and protect the integrity of the epithelial barrier. CLE can be objective, accurate, and real time in investigating gap density. Teprenone and rabeprazole can prevent indomethacin-induced intestinal lesions and protect the epithelial barrier by intervening in the tumor necrosis factor-α pathway. Gap density was expected to be an indicator of evaluating intestinal inflammation and drug efficacy.

Interferon-γ-Induced Intestinal Epithelial Barrier Dysfunction by NF-κB/HIF-1α Pathway

Interferon-γ (IFN-γ) plays an important role in intestinal barrier dysfunction. However, the mechanisms are not fully understood. As hypoxia-inducible factor-1 (HIF-1) is a critical determinant response to hypoxia and inflammation, which has been shown to be deleterious to intestinal barrier function, we hypothesized that IFN-γ induces loss of barrier function through the regulation of HIF-1α activation and function. In this study, we detected the expressions of HIF-1α and tight junction proteins in IFN-γ-treated T84 intestinal epithelial cell line. IFN-γ led to an increase of HIF-1α expression in time- and dose-dependent manners but did not change the expression of HIF-1β. The IFN-γ-induced increase in HIF-1α was associated with an activation of NF-κB. Treatment with the NF-κB inhibitor, pyrolidinedithiocarbamate (PDTC), significantly suppressed the activation of NF-κB and the expression of HIF-1α. In addition, IFN-γ also increased intestinal epithelial permeability and depletion of tight junction proteins; inhibition of NF-κB or HIF-1α prevented the increase in intestinal permeability and alteration in tight junction protein expressions. Interestingly, we demonstrated that a significant portion of IFN-γ activation NF-kB and modulation tight junction expression is mediated through HIF-1α. Taken together, this study suggested that IFN-γ induced the loss of epithelial barrier function and disruption of tight junction proteins, by upregulation of HIF-1α expression through NF-κB pathway.

Intestinal epithelial barrier function in liver cirrhosis: an extensive review of the literature

Recent evidence suggests that translocation of bacteria and bacterial products, such as endotoxin from the intestinal lumen into the systemic circulation is a contributing factor in the pathogenesis of chronic liver diseases and the development of complications in cirrhosis. In addition to alterations in the intestinal microbiota and immune system, dysfunction of the intestinal epithelial barrier may be an important factor facilitating bacterial translocation. This review aims to provide an overview of the current evidence of intestinal epithelial barrier dysfunction in human chronic liver diseases and cirrhosis, and to discuss possible contributing factors and mechanisms. Data suggest the presence of intestinal epithelial barrier dysfunction in patients with chronic liver diseases, but are more convincing in patients with cirrhosis, especially in those with complications. The barrier dysfunction can result from both direct and indirect effects of aetiological factors, such as alcohol and obesity, which can cause chronic liver diseases and ultimately cirrhosis. On the other hand characteristics of cirrhosis itself, including portal hypertension, alterations in the intestinal microbiota, inflammation and oxidative stress can affect barrier function of both small and large intestine and may contribute to the development of complications. In conclusion, there are indications for intestinal epithelial barrier dysfunction in patients with chronic liver diseases and especially in patients with cirrhosis, which can be caused by various factors affecting both the small and large intestine.

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca2+ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.