scholarly journals Chlorothalonil induces the intestinal epithelial barrier dysfunction in Caco-2 cell-based in vitro monolayer model by activating MAPK pathway

2021 ◽  
Author(s):  
Huaping Tao ◽  
Zhiwei Bao ◽  
Zhengwei Fu ◽  
Yuanxiang Jin
Keyword(s):  
Messenger Rna ◽  
Structure And Function ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Intestinal Epithelial Barrier ◽  
Cell Monolayers ◽  
Mitogen Activated Protein ◽  
Exogenous Compounds ◽  
And Function

Abstract The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated messenger RNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the messenger RNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway.

scholarly journals Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism

2013 ◽  
Vol 304 (5) ◽  
pp. G479-G489 ◽  
Author(s):  
Katherine R. Groschwitz ◽  
David Wu ◽  
Heather Osterfeld ◽  
Richard Ahrens ◽  
Simon P. Hogan
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Barrier Function ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Intestinal Epithelial Barrier ◽  
Epithelial Barrier Function ◽  
Epithelial Barrier Dysfunction

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.

scholarly journals Rebeccamycin Attenuates TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production

2017 ◽  
Vol 41 (5) ◽  
pp. 1924-1934 ◽  
Author(s):  
Akihiro Watari ◽  
Yuta Sakamoto ◽  
Kota Hisaie ◽  
Kazuki Iwamoto ◽  
Miho Fueta ◽  
...  
Keyword(s):  
Barrier Function ◽  
Myosin Light Chain ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Intestinal Epithelial Barrier ◽  
Epithelial Barrier Function ◽  
Cell Monolayers ◽  
Tnf Α ◽  
Epithelial Barrier Dysfunction

Background/Aims: Although proinflammatory cytokine–induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. Methods: To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Results: Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Conclusion: Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.

Collagen peptides ameliorate intestinal epithelial barrier dysfunction in immunostimulatory Caco-2 cell monolayers via enhancing tight junctions

2017 ◽  
Vol 8 (3) ◽  
pp. 1144-1151 ◽  
Author(s):  
Qianru Chen ◽  
Oliver Chen ◽  
Isabela M. Martins ◽  
Hu Hou ◽  
Xue Zhao ◽  
...  
Keyword(s):  
Barrier Function ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Intestinal Epithelial Barrier ◽  
Cell Monolayers ◽  
Collagen Peptides ◽  
Epithelial Barrier Dysfunction

Alaska pollock skin derived collagen peptides could be considered as dietary supplements for intestinal barrier function promotion and associated diseases.

Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of occludin, without altering epithelial permeability

2012 ◽  
Vol 302 (3) ◽  
pp. G343-G351 ◽  
Author(s):  
Tamia K. Lapointe ◽  
Andre G. Buret
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Structure And Function ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Barrier Structure ◽  
Intestinal Epithelial Barrier ◽  
Epithelial Barrier Function ◽  
And Function

Compromised epithelial barrier function and tight junction alterations are hallmarks of a number of gastrointestinal disorders, including inflammatory bowel disease (IBD). Increased levels of IL-18 have been observed in mucosal samples from Crohn's disease and ulcerative colitis patients. Remarkably, several reports have demonstrated that immunological or genetic blockage of IL-18 ameliorates the severity of colitis in multiple in vivo models of IBD. Nevertheless, the effects of IL-18 on intestinal epithelial barrier function remain unclear. We hypothesized that IL-18 could disrupt intestinal epithelial barrier structure and function, thus contributing to tissue damage in the context of IBD. The aims of the present study were to determine the effects of IL-18 on epithelial barrier structure and function and to characterize the mechanisms involved in these modulatory properties. Human colonic epithelial Caco-2 monolayers were coincubated with IL-18 for 24 h and processed for immunocytochemistry, immunoblotting, quantitative PCR, and permeability measurements (transepithelial resistance, FITC-dextran fluxes, and bacterial translocation). Our findings indicate that IL-18 selectively disrupts tight junctional occludin, without affecting the distribution pattern of claudin-4, claudin-5, zonula occludens-1, or E-cadherin. This effect coincided with a significant increase in myosin light chain kinase (MLCK) protein levels and activity. Pharmacological inhibition of MLCK and NF-κB prevented IL-18-induced loss of occludin. Although too subtle to alter paracellular permeability, these fine changes correlated with an MLCK-dependent increase in neutrophil transepithelial migration. In conclusion, our data suggest that IL-18 may potentiate inflammation in the context of IBD by facilitating neutrophil transepithelial migration via MLCK-dependent disruption of tight junctional occludin.

scholarly journals Salmonella Effector SpvB Disrupts Intestinal Epithelial Barrier Integrity for Bacterial Translocation

2020 ◽  
Vol 10 ◽  
Author(s):  
Lanqing Sun ◽  
Sidi Yang ◽  
Qifeng Deng ◽  
Kedi Dong ◽  
Yuanyuan Li ◽  
...  
Keyword(s):  
Bacterial Translocation ◽  
Junctional Complex ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Intestinal Epithelial Barrier ◽  
Barrier Integrity ◽  
Epithelial Barrier Dysfunction

Salmonella are common enteric bacterial pathogens that infect both humans and animals. Intestinal epithelial barrier, formed by a single layer of epithelial cells and apical junctional complex (AJC), plays a crucial role in host defense against enteric pathogens to prevent bacterial translocation. However, the underlying mechanisms of intestinal epithelial barrier dysfunction caused by Salmonella are poorly understood. It is found that a locus termed Salmonella plasmid virulence (spv) gene exists extensively in clinically important Salmonella serovars. SpvB is a key effector encoded within this locus, and closely related to Salmonella pathogenicity such as interfering with autophagy and iron homeostasis. To investigate the interaction between SpvB and intestinal epithelial barrier and elucidate the underlying molecular mechanism, we used the typical foodborne disease agent Salmonella enterica serovar Typhimurium (Salmonella typhimurium) carrying spvB or not to construct infection models in vivo and in vitro. C57BL/6 mice were orally challenged with S. typhimurium wild-type strain SL1344 or spvB-deficient mutant strain SL1344-ΔspvB. Caco-2 cell monolayer model, as a widely used model to mimic the human intestinal epithelium in vitro, was infected with SL1344, SL1344-ΔspvB, or spvB complementary strain SL1344-c-ΔspvB, respectively. The results showed that SpvB enhanced bacterial pathogenicity during S. typhimurium infection in vivo, and contributed to intestinal epithelial barrier dysfunction in both infection systems. This SpvB-mediated barrier dysfunction was attributed to the cellular redistribution of Claudin-1, Occludin, and E-cadherin junctional proteins. Moreover, by using pharmacological inhibitors, we found that F-actin rearrangement and suppression of protein kinase C (PKC) signaling pathway were involved in SpvB-mediated barrier dysfunction. In conclusion, the study reveals the contribution of Salmonella effector SpvB to the dysfunction of intestinal epithelial barrier integrity, which facilitates bacterial translocation via the paracellular route to promote Salmonella systemic dissemination. Our findings broaden the understanding of host–pathogen interactions in salmonellosis, and provide new strategies for the therapy in limiting bacterial dissemination during infection.

scholarly journals A Novel Pharmacological Approach to Enhance the Integrity and Accelerate Restitution of the Intestinal Epithelial Barrier

2020 ◽  
Vol 26 (9) ◽  
pp. 1340-1352
Author(s):  
Xuelei Cao ◽  
Lei Sun ◽  
Susana Lechuga ◽  
Nayden G Naydenov ◽  
Alex Feygin ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Cell Monolayers ◽  
Barrier Disruption ◽  
Epithelial Cell Monolayers ◽  
Immunofluorescence Labeling ◽  
E Cadherin

Abstract Background Disruption of the gut barrier is an essential mechanism of inflammatory bowel diseases (IBDs) contributing to the development of mucosal inflammation. A hallmark of barrier disruption is the disassembly of epithelial adherens junctions (AJs) driven by decreased expression of a major AJ protein, E-cadherin. A group of isoxazole compounds, such as E-cadherin-upregulator (ECU) and ML327, were previously shown to stimulate E-cadherin expression in poorly differentiated human cancer cells. This study was designed to examine whether these isoxazole compounds can enhance and protect model intestinal epithelial barriers in vitro. Methods The study was conducted using T84, SK-CO15, and HT-29 human colonic epithelial cell monolayers. Disruption of the epithelial barrier was induced by pro-inflammatory cytokines, tumor necrosis factor-α, and interferon-γ. Barrier integrity and epithelial junction assembly was examined using different permeability assays, immunofluorescence labeling, and confocal microscopy. Epithelial restitution was analyzed using a scratch wound healing assay. Results E-cadherin-upregulator and ML327 treatment of intestinal epithelial cell monolayers resulted in several barrier-protective effects, including reduced steady-state epithelial permeability, inhibition of cytokine-induced barrier disruption and junction disassembly, and acceleration of epithelial wound healing. Surprisingly, these effects were not due to upregulation of E-cadherin expression but were mediated by multiple mechanisms including inhibition of junction protein endocytosis, attenuation of cytokine-induced apoptosis, and activation of promigratory Src and AKT signaling. Conclusions Our data highlight ECU and ML327 as promising compounds for developing new therapeutic strategies to protect the integrity and accelerate the restitution of the intestinal epithelial barrier in IBD and other inflammatory disorders.

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