Intestinal Epithelial Tight Junction Barrier Research Articles

Bifidobacterium bifidum inhibits the TNF-α induced increase in intestinal epithelial tight junction permeability via a TLR-2/TLR-6 and PPAR-γ dependent inhibition of NF-κB p50/p65

Background: Bifidobacterium bifidum (BB) is one of the most widely used probiotic bacterial species and BB has been shown to have a protective effect against intestinal inflammation by an enhancement of the intestinal epithelial tight junction (TJ) barrier. Previous studies from our laboratory showed that a specific BB strain, referred to as BB1, caused a marked enhancement of intestinal epithelial TJ barrier, while strain BB4 had no effect. TNF-α contributes to the development of intestinal inflammation in inflammatory conditions of the gut, in part by increasing intestinal permeability and antigenic penetration. TNF-α induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB p50/p65 activation of myosin light chain kinase (MLCK) gene and MLCK-induced opening of the intestinal TJ barrier. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor known to have anti-inflammatory activity by interfering with NF-κB activation. However, the protective effect of BB1 against TNF-α induced increase in intestinal TJ permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to elucidate the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were the model systems used to assess intestinal TJ permeability. Results: 1) TNF-α caused an increase Caco-2 TJ permeability, as assed by Caco-2 trans-epithelial resistance (TER) and trans-epithelial flux of paracellular marker, FITC-Dextran 10kD. 2) Addition of BB1 (1x108 CFU/ml) to the apical membrane compartment inhibited the TNF-α increase in Caco-2 epithelial TJ permeability, while strain BB4 had no effect. 3) BB1 but not BB4, caused an increase in Caco-2 TLR-2 expression on the apical membrane surface, and siRNA induced knockdown of TLR-2 prevent the BB1 inhibition of TNF-α induced increase in Caco-2 TJ permeability. 4) TNF-α caused a rapid phosphorylation and degradation of Caco-2 IKK-α, as well as and phosphorylation and nuclear translocation of NF-κB p50/p65, indicating NF-κB activation. BB1, but not BB4, inhibited the NF-κB activation. 5) TNF-α activation of NF-κB was associated with an increase in MLCK gene (increase in MLCK promoter activity and mRNA transcription) and kinase activity. BB1, but not BB4, inhibited the TNF-α induced increase in MLCK gene and kinase activity. 6) BB1 but not BB4, caused a rapid PPAR-γ activation (PPAR-γ phosphorylation and nuclear translocation), and siRNA induced knockdown of PPAR-γ inhibited the TNF-α induced activation of NF-κB and increase in Caco-2 TJ permeability. 7) Intraperitoneal injection of TNF-α (5 mg) also caused time-dependent increase in mouse small intestinal permeability (IP) to FITC-Dextran 10kD. TNF-α caused a degradation of IkB-α in intestinal tissue and nuclear translocation of NF-kB in mouse enterocyte. 8) BB1, but not BB4, selectively activated PPAR-γ in mouse enterocytes and inhibited the TNF-α-induced activation of NF-κB, resulting in a decrease in mouse IP. 9) BB1 inhibition of TNF-α activation of mouse enterocyte NF-κB and increase in IP was prevented in mice with intestinal epithelial cell-specific knockout of PPAR-γ Villin-cre and TLR-2 Villin-cre mice. Conclusion: BB1 protects against the TNF-α induced increase in intestinal TJ permeability in a strain-specific manner. BB1 protects against the TNF-α induced increase in intestinal TJ permeability via a novel intracellular mechanism involving BB1/TLR-2 pathway activation of PPAR-γ, and PPAR-γ mediated inhibition of NF-κB p50/p65 and MLCK gene in enterocyte. This research project was supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-121073-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Polystyrene microplastic-induced oxidative stress triggers intestinal barrier dysfunction via the NF-κB/NLRP3/IL-1β/MCLK pathway

Emerging evidence has demonstrated the association between microplastics (MPs) with a diameter of <5 mm and the risk of intestinal diseases. However, the molecular mechanisms contributing to MP-induced intestinal barrier dysfunction have not been fully appreciated. In this study, C57BL/6 J mice were exposed to polystyrene microplastics (PS-MPs, 0.2, 1 or 5 μm) at 1 mg/kg body weight daily by oral gavage for 28 days. We found that PS-MPs exposure induced oxidative stress and inflammatory cell infiltration in mice colon, leading to an increased expression of pro-inflammatory cytokine. Moreover, there were an increase in intestinal permeability and decrease in mucus secretion, accompanied by downregulation of tight junction (TJ)-related zonula occluden-1 (ZO-1), occluding (OCLN) and claudin-1 (CLDN-1) in mice colon. Especially, 5 μm PS-MPs (PS5)-induced intestinal epithelial TJ barrier damage was more severe than 0.2 μm PS-MPs (PS0.2) and 1 μm PS-MPs (PS1). In vitro experiments indicated that PS5-induced oxidative stress upregulated the expression of nuclear factor kappa B (NF-κB), nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome, and myosin light chain kinase (MLCK). Meanwhile, pre-treatment with the antioxidant NAC, NLRP3 inhibitor MCC950 and MLCK inhibitor ML-7 considerably reduced PS5-triggered reactive oxygen species (ROS) production and inflammatory response, inhibited the activation of the NF-κB/NLRP3/MLCK pathway, and upregulated ZO-1, OCLN and CLDN-1 expression in Caco-2 cells. Taken together, our study demonstrated that PS-MPs cause intestinal barrier dysfunction through the ROS-dependent NF-κB/NLRP3/IL-1β/MLCK pathway.

Alpha-tocopherylquinone differentially modulates claudins to enhance intestinal epithelial tight junction barrier via AhR and Nrf2 pathways

Defects in intestinal epithelial tight junctions (TJs) allow paracellular permeation of noxious luminal antigens and are important pathogenic factors in inflammatory bowel disease (IBD). We show that alpha-tocopherylquinone (TQ), a quinone-structured oxidation product of vitamin E, consistently enhances the intestinal TJ barrier by increasing barrier-forming claudin-3 (CLDN3) and reducing channel-forming CLDN2 in Caco-2 cell monolayers (invitro), mouse models (invivo), and surgically resected human colons (exvivo). TQ reduces colonic permeability and ameliorates colitis symptoms in multiple colitis models. TQ, bifunctionally, activates both aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. Genetic deletion studies reveal that TQ-induced AhR activation transcriptionally increases CLDN3 via xenobiotic response element (XRE) in the CLDN3 promoter. Conversely, TQ suppresses CLDN2 expression via Nrf2-mediated STAT3 inhibition. TQ offers a naturally occurring, non-toxic intervention for enhancement of the intestinal TJ barrier and adjunct therapeutics to treat intestinal inflammation.

Bifidobacterium bifidum causes an enhancement of the intestinal epithelial tight junction barrier is mediated by TLR-2 dependent increases of Toll-interacting protein (TOLLIP) expression in a MYD88 independent manner

Background: Bifidobacteria are commonly utilized probiotics that have been shown to protect against intestinal inflammation in part by enhancing the intestinal epithelial tight junction (TJ) barrier function. It has been recently shown that Bifidobacterium bifidum enhances the intestinal epithelial TJ barrier in a strain-specific and toll-like receptor-2 (TLR-2) dependent manner. Toll-interacting protein (TOLLIP) is a potent inhibitory, cytosolic adaptor protein that represses myeloid differentiation primary response 88 (MYD88). However, the association between BB, TLR-2, and TOLLIP in mediating the BB enhancement of intestinal epithelial TJ barrier remains unclear. Aims: The major aim of this study was to delineate the mechanisms that mediate BB enhancement of the intestinal TJ barrier by examining the interaction of BB with the membrane receptor TLR-2 and the potent TLR modulator TOLLIP in the intestinal epithelial cells. Methods: An in-vitro intestinal epithelial model system consisting of filter-grown Caco-2 monolayers was used, and various biochemical methods and confocal microscopy were applied. Results: 1) A specific strain of BB (1x108 CFU/ml) referred to as BB1 caused a marked increase in Caco-2 transepithelial resistance (TER) (80% increase) and a decrease in trans-epithelial flux of paracellular marker. Other BB strains examined (five total strains including BB4) caused a modest increase to (25%-1%) no changes in Caco-2 TER, suggesting that BB enhancement of Caco-2 TJ barrier function was strain-specific. Strain BB4 had no effect on Caco-2 TJ barrier function. 2) BB1 but not BB4 caused an increase in TLR-2 expression. 3) BB1 induced increase of TLR-2 expression was independent of MYD88, and MYD88 was not required for the BB1-induced enhancement of intestinal TJ barrier. 4) BB1, not BB4, caused an activation of TOLLIP expression 4) The siRNA-induced knockdown of TLR-2 prevented the BB1-induced increase in TOLLIP expression and recruitments to apical membrane junctions, and also prevented the BB1 induced increase in Caco-2 TER and drop in inulin flux. 5) The siRNA-induced knockdown of TOLLIP prevented the BB1-induced increase in Caco-2 TER and drop in inulin flux, and inhibition of IRAK-1 kinase activity. Conclusion: These studies provide a novel insight into the mechanisms that mediate BB1 enhancement and protection of intestinal TJ barrier; our data show for the first time that TLR-2/TOLLIP plays an integral role in BB1 enhancement of TJ barrier function. The BB-induced enhancement of the intestinal epithelial TJ barrier was mediated by TLR-2 dependent signaling recruitment of TOLLIP, and BB enhancement of the TJ barrier was blocked by targeted knockdown of TLR-2 and TOLLIP. TOLLIP represent a potential novel therapeutic target for IBD. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lactobacillus acidophilus Induces Enhancement of the Intestinal Epithelial Tight Junction Barrier Function by Toll-like Receptor-2 Independent of MyD88

A defective intestinal epithelial tight junction (TJ) barrier has been postulated to be a key pathogenic factor in allergic disorders, including food allergy. Enhancement of the TJ barrier might be a target for prevention or treatment of such diseases. Our lab recently showed that Lactobacillus acidophilus (LA) caused an enhancement of the intestinal TJ barrier that was mediated by attachment of live LA to toll-like receptor-2 (TLR2) on the apical membrane surface of enterocytes. The aim of this study was to delineate TLR2 signaling mechanisms mediating the LA-induced enhancement of the intestinal TJ barrier.

Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux.

Functional loss of the gut epithelium's paracellular tight junction [TJ] barrier and defective autophagy are factors potentiating inflammatory bowel disease [IBD]. Previously, we showed the role of autophagy in enhancing the intestinal TJ barrier via pore-forming claudin-2 degradation. How autophagy regulates the TJ barrier-forming proteins remains unknown. Here, we investigated the role of autophagy in the regulation of occludin, a principal TJ component involved in TJ barrier enhancement. Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in intestinal epithelial cells, mouse colonocytes and human colonoids. Autophagy induction enriched membrane occludin levels and reduced paracellular permeability of macromolecules. Autophagy-mediated TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier-enhancing effect against macromolecular flux. Autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss. Autophagy enhanced the phosphorylation of ERK-1/2 and inhibition of these kinases in Caco-2 cells and human colonic mucosa prevented the macromolecular barrier-enhancing effects of autophagy. In vivo, autophagy induction by rapamycin enhanced occludin levels in wild-type mouse intestines and protected against lipopolysaccharide- and tumour necrosis factor-α-induced TJ barrier loss. Disruption of autophagy with acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ permeability and exacerbating the effect of experimental colitis. Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 mitogen-activated protein kinase-dependent mechanism.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Lactobacillus acidophilus causes enhancement of the intestinal tight junction barrier by a toll-like receptor-2-dependent increase in occludin

A defective intestinal epithelial tight junction (TJ) barrier has been implicated as an important pathogenic factor in various gastrointestinal immune disorders, including celiac disease and food allergy. Enhancement of the intestinal TJ barrier might be an important therapeutic target for prevention or treatment of such diseases. Recent studies from our lab showed that Lactobacillus acidophilus (LA), a naturally occurring probiotic bacteria, caused a marked and unique enhancement of the intestinal TJ barrier mediated by toll-like receptor-2 (TLR2).

The functional role of miRNAs in inflammatory pathways associated with intestinal epithelial tight junction barrier regulation in IBD

Abstract Inflammatory bowel disease – Crohn's disease and ulcerative colitis – is an immune-mediated chronic disorder with still not fully elucidated complex mechanisms of pathogenesis and pathophysiology. Intestinal epithelial barrier (IEB) dysregulation is one of the major underlying mechanisms of inflammatory process induction in IBD. Proper IEB integrity is maintained to a large extent by intercellular tight junctions, the function of which can be modified by many molecules, including miRNAs. MiRNAs belong to noncoding and non-messenger RNAs, which can modulate gene expression by binding predicted mRNAs. In this review, we summarize and discuss the potential role of miRNAs in the regulation of inflammatory signaling pathways affecting the function of the intestinal epithelial barrier in IBD, with particular emphasis on therapeutic potentials. The aim of the review is also to determine the further development directions of the studies on miRNA in the modulation of the intestinal epithelial barrier in IBD.

IL-1β and the Intestinal Epithelial Tight Junction Barrier.

The intestinal epithelial tight junction (TJ) barrier controls the paracellular permeation of contents from the intestinal lumen into the intestinal tissue and systemic circulation. A defective intestinal TJ barrier has been implicated as an important pathogenic factor in inflammatory diseases of the gut including Crohn’s disease, ulcerative colitis, necrotizing enterocolitis, and celiac disease. Previous studies have shown that pro-inflammatory cytokines, which are produced during intestinal inflammation, including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ, have important intestinal TJ barrier-modulating actions. Recent studies have shown that the IL-1β-induced increase in intestinal TJ permeability is an important contributing factor of intestinal inflammation. The IL-1β-induced increase in intestinal TJ permeability is mediated by regulatory signaling pathways and activation of nuclear transcription factor nuclear factor-κB, myosin light chain kinase gene activation, and post-transcriptional occludin gene modulation by microRNA and contributes to the intestinal inflammatory process. In this review, the regulatory role of IL-1β on intestinal TJ barrier, the intracellular mechanisms that mediate the IL-1β modulation of intestinal TJ permeability, and the potential therapeutic targeting of the TJ barrier are discussed.

Bifidobacterium bifidum Enhances the Intestinal Epithelial Tight Junction Barrier and Protects against Intestinal Inflammation by Targeting the Toll-like Receptor-2 Pathway in an NF-κB-Independent Manner.

Defective intestinal tight junction (TJ) barrier is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). To date, there are no effective therapies that specifically target the intestinal TJ barrier. Among the various probiotic bacteria, Bifidobacterium, is one of the most widely studied to have beneficial effects on the intestinal TJ barrier. The main purpose of this study was to identify Bifidobacterium species that cause a sustained enhancement in the intestinal epithelial TJ barrier and can be used therapeutically to target the intestinal TJ barrier and to protect against or treat intestinal inflammation. Our results showed that Bifidobacterium bifidum caused a marked, sustained enhancement in the intestinal TJ barrier in Caco-2 monolayers. The Bifidobacterium bifidum effect on TJ barrier was strain-specific, and only the strain designated as BB1 caused a maximal enhancement in TJ barrier function. The mechanism of BB1 enhancement of intestinal TJ barrier required live bacterial cell/enterocyte interaction and was mediated by the BB1 attachment to Toll-like receptor-2 (TLR-2) at the apical membrane surface. The BB1 enhancement of the intestinal epithelial TJ barrier function was mediated by the activation of the p38 kinase pathway, but not the NF-κB signaling pathway. Moreover, the BB1 caused a marked enhancement in mouse intestinal TJ barrier in a TLR-2-dependent manner and protected against dextran sodium sulfate (DSS)-induced increase in mouse colonic permeability, and treated the DSS-induced colitis in a TJ barrier-dependent manner. These studies show that probiotic bacteria BB1 causes a strain-specific enhancement of the intestinal TJ barrier through a novel mechanism involving BB1 attachment to the enterocyte TLR-2 receptor complex and activation of p38 kinase pathway.

Matrix Metalloproteinase-9 (MMP-9) induced disruption of intestinal epithelial tight junction barrier is mediated by NF-κB activation.

BackgroundMatrix Metalloproteinase-9 (MMP-9) has been shown to play a key role in mediating inflammation and tissue damage in inflammatory bowel disease (IBD). In patients with IBD, the intestinal tight junction (TJ) barrier is compromised as characterized by an increase in intestinal permeability. MMP-9 is elevated in intestinal tissue, serum and stool of patients with IBD. Previous studies from our laboratory showed that MMP-9 causes an increase in intestinal epithelial TJ permeability and that the MMP-9 induced increase in intestinal permeability is an important pathogenic factor contributing to the development of intestinal inflammation in IBD. However, the intracellular mechanisms that mediate the MMP-9 modulation of intestinal barrier function remain unclear.AimsThe main aim of this study was to further elucidate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ permeability using Caco-2 monolayers as an in-vitro model system.ResultsMMP-9 induced increase in Caco-2 TJ permeability was associated with activation and cytoplasmic-to-nuclear translocation of NF-κB p65. Knocking-down NF-κB p65 by siRNA transfection prevented the MMP-9 induced expression of the NF-κB target gene IL-8, myosin light chain kinase (MLCK) protein expression, and subsequently prevented the increase in Caco-2 TJ permeability. In addition, the effect of MMP-9 on Caco-2 intestinal epithelial TJ barrier function was not mediated by apoptosis or necrosis.ConclusionOur data show that the MMP-9 induced disruption of Caco-2 intestinal epithelial TJ barrier function is regulated by NF-κB pathway activation of MLCK.

PROTON PUMP INHIBITORS (PPI) INDUCES COLONIC TIGHT JUNCTION BARRIER (TJ) DYSFUNCTION VIA AN UPREGULATION OF TJ PORE FORMING CALUDIN-2 PROTEIN

Abstract Background Proton pump inhibitors (PPIs) are highly effective antagonists of gastric acid secretion and are widely used to treat a number of gastroesophageal disorders, including peptic ulcer disease, GERD, and Barrett’s esophagus. PPI-induced elevation in intra-gastric pH and subsequent alterations of gastrointestinal physiology are thought to cause undesired effects on the entire GI tract. Defective intestinal Tight Junction (TJ) barrier is an important pathogenic factor for intestinal inflammation. Claudin-2 is a pore forming TJ protein whose overexpression causes selective increase in TJ permeability to small molecules. Claudin-2 expression is known to be up-regulated in intestinal inflammation and inflammation-associated colon carcinogenesis. The effect of PPI on Intestinal TJ barrier is not known. Aim: The aim of the present study was to study the effect of PPI on Intestinal TJ permeability. Methods A cell culture model of filter grown human intestinal epithelial Caco-2 monolayers, was used to study intestinal epithelial TJ barrier function. The mouse colonic permeability was measured by Ussing chambers studies. Western blot (WB) and Immunofluorescence (IF) was used to study the protein expression of claudin-2 in Caco-2 cells and in mouse colon. Cell surface biotinylation was used to study intracellular trafficking of TJ proteins. Results PPIs, caused a concentration- and time-dependent decrease in transepithelial resistance (TER), and increase in urea flux in filter-grown Caco-2 cells. Further studies on intestinal TJ revealed that PPI caused a significant increase in pore forming TJ protein claudin-2 protein level (western blot), but not mRNA (RT-PCR) in Caco-2 monolayers. Claudin-2 was co-localized to its known intercellular trafficking vesicles, clathrin pits. PPI treatment caused an increase in expression and junctional localization of claudin-2 and a decrease in its cytoplasmic co-localization with clathrin. Similarly, pulse-chase assay showed that the claudin-2 half-life was increased by PPI. In cell surface biotinylation assay, the rate of claudin-2 endocytosis was significantly reduced by PPI. In ex-vivo, Ussing chamber studies, PPI administration for 45 days resulted in a significant decrease in murine colonic TER and increase in urea flux. Immunofluorescence and Western blot analysis showed that PPI also caused an increase in TJ protein claudin-2 expression in mice colonic mucosa. Conclusion Our results suggest that the PPIs induces colonic TJ permeability via increasing pore forming claudin-2 levels. PPIs affect intracellular trafficking to reduce claudin-2 degradation and increased its junctional localization.

Delivery of polysaccharides from Ophiopogon japonicus (OJPs) using OJPs/chitosan/whey protein co-assembled nanoparticles to treat defective intestinal epithelial tight junction barrier

The polysaccharides from Ophiopogon japonicus (OJPs) were known to have protective effects against diabetes, and cardiovascular and chronic inflammatory diseases. However, OJPs were poorly absorbed after oral administration, resulting in limited efficacy because of the low bioavailability. In this study, OJPs extracted and fractionated from Ophiopogon japonicus were used to prepare OJPs/chitosan (CS)/whey protein (WP) co-assembled nanoparticles. The OJPs/CS/WP nanoparticles showed high biocompatibility and inhibited the cytotoxicity of RAW264.7 cells induced by nickel. With the assistance of CS and WP, the anti-inflammatory and antioxidant activities of OJPs were enhanced because the nanoparticles improved OJPs uptake by RAW264.7 macrophage cells as evidenced by efficient scavenging of DPPH and ABTS free radicals and effective inhibition of NO production and the gene expressions of iNOS, COX2, TNF-α, CCL2, and CXCL2 inflammatory signals. Determining the transepithelial electrical resistance and paracellular permeability of Caco-2 monolayer/macrophage co-cultured system suggested that the OJPs-loaded nanoparticles effectively protected the intestinal epithelial barrier integrity against the damage caused by LPS-stimulated macrophage inflammation and attenuated the defects of intestinal epithelial TJ barrier and permeability. These findings suggest that the OJPs/CS/WP nanoparticles may be potential carriers for oral delivery of OJPs to treat intestinal barrier defects, such as inflammatory bowel disease (IBD).

Protective effect of emodin on intestinal epithelial tight junction barrier integrity in rats with sepsis induced by cecal ligation and puncture.

The present study investigated the protective effects of emodin on intestinal epithelial tight junction (TJ) barrier integrity in cecal ligation and puncture (CLP)-induced septic rats and its possible mechanisms of action. Healthy male Sprague-Dawley rats were randomly divided into three groups (n=20 per group): Sham group, CLP group and CLP + emodin group. Animals were sacrificed at 12 and 24 h after the model was established. Abdominal aortic blood and specimens of the ileum were harvested for analysis. The histopathological changes in intestinal mucosa and the ultrastructures of intestinal epithelial cells were investigated using light microscopy and transmission electron microscopy. The integrity of the intestinal barrier was assessed by examining plasma diamine oxidase (DAO) levels and the ratio of urine lactulose to mannitol (L/M). The levels of the intestinal TJ proteins claudin-3, zonula occludens (ZO)-1 and occludin were detected using immunohistochemistry, western blotting and reverse transcription-quantitative PCR. The results showed that the pathological damage to intestinal mucosa and the intestinal tissue injury score in the CLP + emodin group were significantly reduced compared to those of the CLP group, and the differences were more obvious at 24 h compared with 12 h. DAO activity and the L/M ratio in the emodin pre-treatment group decreased significantly at 24 h compared with the CLP groups. The protein and mRNA levels of the TJ proteins claudin-3, ZO-1 and occludin in the emodin pre-treatment groups at 12 and 24 h were increased, while occludin mRNA level was found to be decreased compared with the CLP groups. The present study suggested that emodin may significantly reduce the damage to the intestinal epithelial barrier in sepsis, inhibit intestinal barrier permeability and protect intestinal barrier integrity. Emodin may protect intestinal barrier integrity by elevating expression levels of the TJ proteins claudin-3, ZO-1 and occludin in CLP rats.

Flavonoids modulate tight junction barrier functions in hyperglycemic human intestinal Caco-2 cells.

Diabetes mellitus is a chronic disease requiring lifelong medical attention. With hundreds of millions suffering worldwide and a rapidly rising incidence, diabetes mellitus poses a great burden on health care systems. Recent studies investigating the underlying mechanisms involved in disease development in diabetes point to the role of the dysregulation of the intestinal barrier. Hyperglycemia-mediated tight junction deformity is known to contribute to leaky gut in various metabolic disorders. The present study aimed to investigate the role of oxidative stress on intestinal epithelial tight junction (TJ) barrier functions in hyperglycemia. Because many flavonoids are known to influence the cellular redox state, exploring these flavonoids may help to understand the role of TJ barrier in hyperglycemia-mediated oxidative stress, which in turn might unfold the association of oxidative stress and dysfunction of barrier-forming TJs. Caco-2 cells were stimulated with high glucose (HG), with or without flavonoids (quercetin, morin, naringenin), for 24 h. We determined cellular viability, levels of reactive oxygen species, and mitochondrial membrane potential in flavonoids treated HG-Caco-2 cells. The levels of the proinflammatory cytokines, glucose uptake, and expression of glucose transporters were determined on flavonoids treatment. We investigated the effect of flavonoids on TJs functions by measuring transepithelial electrical resistance (a TJ integrity marker), membrane permeability using tracer compounds, and the expressions levels of TJs related molecules on hyperglycemic Caco-2 cell monolayers. We found that high glucose treatment resulted in reduced cell viability, increased reactive oxygen species production, measurable mitochondrial dysfunction, and decreased transepithelial electrical resistance, with increased membrane permeability. Treatment with the test flavonoids produced increased cell viability and reduced glucose uptake of HG-Caco-2 cells. A concomitant decrease in reactive oxygen species production, proinflammatory cytokines, and Glut-associated genes and proteins were identified with flavonoid treatment. Flavonoids prevented derangement of TJs protein interaction and stabilized membrane permeability. These findings indicate that flavonoids confer protection against hyperglycemia-mediated oxidative stress and enhance intestinal barrier functions by modulating underlying intracellular molecular mechanisms.

Hydrogen-rich saline protects intestinal epithelial tight junction barrier in rats with intestinal ischemia–reperfusion injury by inhibiting endoplasmic reticulum stress-induced apoptosis pathway

AimTo investigate the effects of hydrogen-rich saline (HRS) on intestinal epithelial tight junction (TJ) barrier in rats with intestinal ischemia–reperfusion injury (IIRI). Materials and methodsThirty-two healthy male Sprague–Dawley (SD) rats were randomly divided into four groups (n = 8 each): Sham group, I/R group, HRS group and 4-PBA group. After 45 min of ischemia and 6 h of reperfusion, the rats were sacrificed to collect serum and ileum for detection. Hematoxylin and eosin (H&E) staining was used to observe the morphology of small intestine. The serum expression levels of intestinal fatty acid binding protein (IFABP), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were determined by enzyme linked immunosorbent assay (ELISA). Imunohistochemistry, immunofluorescence and Western blot were used to detect key proteins in intestinal epithelial TJs, ERS, and ERS-induced apoptosis, including occludin, zonula occludens-1 (ZO-1), glucose-regulated protein 78 (GRP78), X-box binding protein-1 (XBP1), C/EBP-homologous protein (CHOP) and caspase-3. Data was presented as mean ± SEM and compared using one-way ANOVA. A p-value <0.05 was considered significant. ResultsCompared with rats in the I/R group, the Chiu score of ileum damage in the HRS group and 4-PBA group were lower. The levels of serum IFABP, TNF-α, and IL-1β were statistically significant among the groups. Increased expression of TJ proteins occludin and ZO-1 by reducing various parameters of ERS and ERS-induced apoptosis evidenced by down-regulation of the protein levels of GRP78, XBP1, CHOP and caspase-3 were shown in the HRS and 4-PBA groups. ConclusionHRS had potential protective effects on intestinal barrier in IIRI rats. This study suggested that inhibition of excessive ERS and ERS-induced apoptosis by HRS may reduce intestinal epithelial cells damage and maintain the integrity of intestinal epithelial TJ barrier in rats with IIRI.

Significance and dietary regulation of intestinal epithelial tight junction barrier

Significance and dietary regulation of intestinal epithelial tight junction barrier

Antibiotics induced intestinal tight junction barrier dysfunction is associated with microbiota dysbiosis, activated NLRP3 inflammasome and autophagy.

Tight junction barrier is critical to intestinal homeostasis. Applying antibiotics to treat infections is common in clinical practice, which may affect intestinal microbiota. Intestinal microbiota dysbiosis is involved in the occurrence of some gastrointestinal diseases. Therefore, this study was aimed to investigate the influence of antibiotics on intestinal tight junction barrier and the possible underlying mechanisms. Healthy adult female C57BL/6 mice were treated with a broad-spectrum antibiotic cocktail for 14 days. 16S rDNA Illumina sequencing and headspace gas chromatography-mass spectrometry (HS-GC/MS) were respectively used to analyze microbial community and to detect short-chain fatty acids (SCFAs) contents. In vivo intestinal paracellular permeability to fluorescein isothiocyanate-dextran (FITC-dextran) was measured. Protein expression was determined by immunoblotting. Immunofluoresence was applied to observe the distributions of ZO-1, LC3B and ASC. Antibiotics remarkably altered intestinal microbiota composition in healthy mice, accompanying reduced SCFAs’ concentrations. In addition, the intestinal tight junction barrier was disrupted by antibiotic treatment, as evidenced by increased intestinal paracellular permeability to FITC-dextran, decreased tight junction protein expressions, and disrupted ZO-1 morphology. Furthermore, NLRP3 inflammasome and autophagy were activated by antibiotic treatment. In conclusion, intestinal epithelial tight junction barrier dysfunction induced by antibiotics is associated with intestinal microbiota dysbiosis, activated NLRP3 inflammasome and autophagy in mice.