Reduction In Transepithelial Electrical Resistance Research Articles

High-fat diet enhances cell proliferation and compromises intestinal permeability in a translational canine intestinal organoid model

BackgroundEmerging evidence underscores the responsiveness of the mammalian intestine to dietary cues, notably through the involvement of LGR5 + intestinal stem cells in orchestrating responses to diet-driven signals. However, the effects of high-fat diet (HFD) on these cellular dynamics and their impact on gut integrity remain insufficiently understood. Our study aims to assess the multifaceted interactions between palmitic acid (PA), cell proliferation, and the intestinal epithelial barrier using a canine colonoid model. Canine models, due to their relevance in simulating human intestinal diseases, offer a unique platform to explore the molecular mechanisms underlying HFD derived intestinal dysfunction.ResultsCanine colonoids were subjected to PA exposure, a surrogate for the effects of HFD. This intervention revealed a remarkable augmentation of cell proliferative activity. Furthermore, we observed a parallel reduction in transepithelial electrical resistance (TEER), indicating altered epithelium barrier integrity. While E-cadherin exhibited consistency, ZO-1 displayed a noteworthy reduction in fluorescence intensity within the PA-exposed group.ConclusionsBy employing canine intestinal organoid systems, we provide compelling insights into the impact of PA on intestinal physiology. These findings underscore the importance of considering both cell proliferative activity and epithelial integrity in comprehending the repercussions of HFDs on intestinal health. Our study contributes to a deeper understanding of the consequences of HFD on intestinal homeostasis, utilizing valuable translational in vitro models derived from dogs.

Abstract 2125: The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity

Abstract Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of antineoplastic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial. Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, dog and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo. The toxic effects of antineoplastic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs. To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, increased FITC-Dextran permeability and analysis of TJ proteins by immunofluorescence. Furthermore, effects on cell death were assessed by measuring the levels of Lactate Dehydrogenase (LDH) released into the medium by dying cells. In conclusion, our models provide an innovative in vitro solution to further study the toxic effects of drugs on the normal small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered antineoplastic drugs. The model can be considered more representative of the normal intestine than transformed cell lines such as CaCo2. Citation Format: Valentina Ubertini, Sarah Hall, Frida Ponthan, James Wilson. The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2125.

COPD basal cells are primed towards secretory to multiciliated cell imbalance driving increased resilience to environmental stressors

IntroductionEnvironmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised.MethodsWe delineated...

3D MORPHOGENESIS AND BACTERIAL CO-CULTURE IN A CANINE GUT-ON-A-CHIP USING BIOPSY-DERIVED INTESTINAL ORGANOIDS FROM HEALTHY AND IBD PATIENTS

Abstract The understanding of the role of intestinal bacteria in various intestinal diseases has been limited due to the lack of suitable in vitro models. However, recent advancements in Gut-on-a-Chip culture technology provide a promising avenue for exploring complex interactions between intestinal epithelium and bacteria. In this study, we aimed to create a microfluidic Gut-on-a-Chip co-culture system using dogs as a model. Dogs share similarities with humans in terms of intestinal disease pathophysiology, clinical presentations, and intestinal microbiome, making them a relevant model for various intestinal diseases, including inflammatory bowel disease (IBD). We cultured colonoids from both healthy dogs and dogs with IBD on the Gut-on-a-Chip platform to create villus-like structures under dynamic conditions. Co-cultures with non-pathogenic Escherichia coli (NPE) were established in the Gut-on-a-Chip with healthy colonoids, and we conducted a static Transwell (TW) co-culture for comparison. To assess epithelial barrier integrity, we measured transepithelial electrical resistance (TEER), and we evaluated the expression of ZO-1, a tight junction protein, through immunofluorescence staining (IF). Following 6-9 days of uninterrupted medium flow in the Gut-on-a-Chip models, we observed sporadic clusters of villus-like structures (i.e., 3D morphogenesis) in canine intestinal epithelial cells in both the models derived from healthy dogs and those with IBD. The IF confirmed the presence of ZO-1, indicating the establishment of tight junctions. Infection with NPE in the TW system resulted in a significant reduction in TEER (48.2±4.4% at 12 hours and 7.3±5.3% at 24 hours). In contrast, the Gut-on-a-Chip maintained TEER levels at 100.3±12.8% at 12 hours, 94.9±2.2% at 24 hours, and 88.8±15.0% at 48 hours, preserving the 3D structure. The innovative canine Gut-on-a-Chip model, capable of reproducing villus-like structures and preserving a 3D configuration using both healthy and diseased organoids (specifically, IBD), presents a valuable platform for investigating bacterial-epithelium interactions in both human and veterinary medicine. This dynamic microfluidic culture system enables the co-cultivation of living bacteria while maintaining epithelial integrity, facilitating the study of intricate interactions between intestinal bacteria and epithelial crosstalk.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins.

Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin's effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

Mercaptonicotinic acid activated thiolated chitosan (MNA-TG-chitosan) to enable peptide oral delivery by opening cell tight junctions and enhancing transepithelial transport

Recent advances in peptide delivery and nanotechnology has resulted in emergence of several non-parenteral administration routes that replace subcutaneous injections associated with patient discomfort. Thiolated biopolymers are relatively new materials being explored to enhance mucoadhesivity and permeability in these efforts, yet their pH dependent reactivity remains an obstacle. This work focussed on improving the mucoadhesivity of thiolated chitosans by activating them with mercaptonicotinic acid, in a bid to create a novel thiomerized chitosan that can open cell tight junctions for application in oral delivery. The synthesized mercaptonicotinic acid activated thiolated chistoan (MNA-TG-chitosan), along with thiolated chitosan (TG-chitosan) and unmodified chitosan were then used to create insulin nanoparticles (insNPs) using spray drying encapsulation process. Use of MNA-TG-chitosan in place of chitosan resulted in reduction of particle size of insNPs from 318 to 277 nm with no significant changes in polydispersity index (~ 0.2), encapsulation efficiency (~ 99%), insulin loading content (~ 25%) and morphology. Results from in-vitro cytotoxicity on TR146, CaCo2 and HepG2 cell lines revealed no significant effects on cell viability at 50–1000 μg/mL concentration. insNPs encapsulated with the new material, MNA-TG-chitosan, resulted in a 1.5-fold and 4.4-fold higher cellular uptake by HepG2 liver cells where insulin is metabolized, approximately 40% and 600% greater insulin transport through TR146 buccal cell monolayers, and 40% and 150% greater apparent permeability than insNPs encapsulated with unmodified chitosan and TG-chitosan respectively. The higher permeation achieved on using MNA-TG chitosan was attributed to the greater opening of the cell tight junction evidenced by reduction of transepithelial electrical resistance of TR146 buccal cell monolayers. This study demonstrates MNA-TG-chitosan as a promising material for improved peptide oral delivery.

Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction.

Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function. Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

Beneficial Effect of Sustainable Magnetic Treatment of Tap Water Investigations with Cultivated Intestinal Epithelial Cells

(Electro)magnetic fields have been used as a tap water treatment since more than 60 years to prevent water pipe walls from the unwanted deposition of calcium carbonate. In the meantime a lot of research has been done on the effects of magnetic or electromagnetic treatment of water. Most of them deal with physiochemical aspects and only a few report on biological effects on prokaryotes or cultured mammalian cells. Intestinal health plays a key role in respect to systemic health and the intestinal epithelial barrier comes directly into contact with drinking water, the effect of magnetic tap water treatment on the integrity of the intestinal epithelial barrier was investigated by measurement of the transepithelial electrical resistance (TEER). Moreover, the sensitivity of the intestinal epithelial barrier against oxidative stress was investigated. Since the regeneration process of intestinal epithelial cells is essential for the reconstitution and maintenance of the barrier integrity after injury or traumatization in vivo, this aspect was also examined with water ± magnetic treatment. The animal cell line IPEC-J2 was used for the investigations. Local tap water (= initial source water) was obtained before and after magnetic treatment with water core magnets which show a very weak field strength with high magnetic gradients as a special characteristic. TEER increased by 28.5 ± 6.8% after incubation with the tap water with magnetic treatment when compared to the initial tap water (mean value ± standard deviation; p ≤ 0.01 by the two-tailed Wilcoxon-Mann-Whitney rank sum test). Measurement of TEER after oxidative stress showed no marked difference between both tap water samples ± magnetic treatment. In both experimental series there was a significant reduction in TEER by more than 90 % representing a leaky intestinal epithelial cell layer due to oxidative stress. Despite this fact the number of cells in the initial tap water sample was largely decreased whereas the cell density in the treated water samples was much higher. The magnetic treatment of the tap water improved the regeneration of the intestinal epithelial cells by 17.4 ± 7.2% (mean value ± standard deviation; p ≤ 0.05 by the two-tailed Wilcoxon-Mann-Whitney rank sum test) when compared with the initial tap water. The results of the study demonstrate that the water core magnets as used here are able to influence tap water and, subsequently, induce beneficial cellular effects on cultured intestinal epithelial cells. Thus, the tap water with magnetic treatment might be able to promote and maintain intestinal and systemic health in vivo.

Quaternization of high molecular weight chitosan for increasing intestinal drug absorption using Caco-2 cells as an in vitro intestinal model

Potential use of a quaternized chitosan (MW 600 kDa) with 65% of 3-chloro-2-hydroxypropyltrimethylammonium (600-HPTChC65) as an absorptive enhancer was investigated in Caco-2 monolayers. 600-HPTChC65 (0.005% w/v) quickly reduced transepithelial electrical resistance (TEER) to the maximum level in 40 min with full recovery within 6 h after removal. Its TEER reduction was corresponded to increased FD4 transport across the monolayers and disrupted localization of tight junction proteins ZO-1 and occludin at the cell borders. 600-HPTChC65 was densely localized at the membrane surface and intercellular junctions. This chitosan (0.08–0.32% w/v) reduced the efflux ratio of [3H]-digoxin by 1.7- 2 folds, suggesting an increased [3H]-digoxin transport across the monolayers. Its binding with P-gp on Caco-2 monolayer increased the signal of fluorescence-labeled anti-P-gp (UIC2) reactivity due to conformational change. 600-HPTChC65 (0.32% w/v) had no effect on P-gp expression in the Caco-2 monolayers. These results suggest that 600-HPTChC65 could enhance drug absorption through tight junction opening and decreased P-gp function. Its interaction with the absorptive barrier mainly resulted in disrupting ZO-1 and occludin organization as well as changing in P-gp conformation.

Abstract 2754: Transwell-grown monolayers derived from small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs

Abstract Although oral dosing is the most convenient route of drug administration, most anti-neoplastic drugs are administered intravenously due to poor bioavailability because of the epithelial barriers. A major organ involved in oral drug absorption is the small intestine where enterocytes are linked together by tight junctions (TJs). TJs regulate paracellular transport by creating a selectively permeable barrier to small molecules, ions, and water, thereby restricting the uptake of drugs. Transient modulation of the TJs is considered a potential strategy to improve drug delivery. Human small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. However, their geometry prevents access to the apical compartment of the epithelium (the outer, or luminal, surface being located within the organoid with the basal surface on the outside), making them unsuitable for the study of orally-administered drugs. Here we describe a method to generate transwell-grown cell monolayers derived from normal human small intestine, that allows direct access to the apical surface. We demonstrate that the format provides an in vivo-like cell composition, comprising of all the intestinal cellular types, including stem cells, Paneth cells, goblet cells, enteroendocrine cells and enterocytes, and respond to treatment with ‘tool drug’ TJ modulators such as sodium caprate, chitosan, EGTA and various cytokines. Briefly, for the generation of transwell-grown cell monolayers, SI organoids were dissociated into single cells and plated in matrix-coated transwell inserts. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements. Treatments with TJ modulators were applied after TEER reached a plateau (8-10 days after plating), and effects on TJs were evaluated 24-72 hours after treatment by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. In conclusion, our models provide an innovative in vitro solution to study the effect of TJ modulators on increasing paracellular transport of orally-administered, anti-neoplastic drugs. Furthermore, as anti-neoplastic drugs can also have toxic effects on the intestinal epithelium by affecting TJs, thereby creating a leaky barrier, the model is useful to predict such effects and stratify drug candidates prior to in vivo studies. The model also provides a system to test the restorative potential of molecules on barrier integrity that could be used in combination or after the administration of anti-neoplastic drugs. Citation Format: Valentina Ubertini, Frida Ponthan, Aude-Marine Bonavita, James Wilson. Transwell-grown monolayers derived from small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2754.

IE-DAP Induced Inflammatory Response and Tight Junction Disruption in Bovine Mammary Epithelial Cells via NOD1-Dependent NF-κB and MLCK Signaling Pathway.

γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a bacterial cell wall component, can trigger an inflammatory response. A mammary inflammatory response causes tight junction (TJ) dysfunction. This study aimed to explore the effects and involved mechanisms of iE-DAP-induced inflammatory response on the TJ integrity in bovine mammary epithelial cells (BMECs). The results showed that iE-DAP-induced inflammatory response and TJ disruption was associated with increased expression levels of inflammatory cytokines and decreased gene expression of ZO-1 and Occludin, as well as a reduction in transepithelial electrical resistance and elevation in paracellular dextran passage. While MLCK inhibitor ML-7 reversed the TJ disruption induced by iE-DAP. NF-κB inhibitor BAY 11-7085 hindered the activation of NF-κB and MLCK signaling pathways, the inflammatory response and TJ disruption induced by iE-DAP. NOD1-specific shRNA also inhibited the activation of the NOD1/NF-κB signaling pathway and reversed the inflammatory response and TJ injury in iE-DAP-treated BMECs. Above results suggest that iE-DAP activated the NF-κB and MLCK signaling pathway in NOD1-dependent manner, which promoted the transcription of inflammatory cytokines and altered the expression and distribution of tight junction proteins, finally caused inflammatory response and TJ disruption. This study might provide theoretical basis and scientific support for the prevention and treatment of mastitis.

Does Deoxynivalenol Affect Amoxicillin and Doxycycline Absorption in the Gastrointestinal Tract? Ex Vivo Study on Swine Jejunum Mucosa Explants.

The presence of deoxynivalenol (DON) in feed may increase intestinal barrier permeability. Disturbance of the intestinal barrier integrity may affect the absorption of antibiotics used in animals. Since the bioavailability of orally administered antibiotics significantly affects their efficacy and safety, it was decided to evaluate how DON influences the absorption of the most commonly used antibiotics in pigs, i.e., amoxicillin (AMX) and doxycycline (DOX). The studies were conducted using jejunal explants from adult pigs. Explants were incubated in Ussing chambers, in which a buffer containing DON (30 µg/mL), AMX (50 µg/mL), DOX (30 µg/mL), a combination of AMX + DON, or a combination of DOX + DON was used. Changes in transepithelial electrical resistance (TEER), the flux of transcellular and intracellular transport markers, and the flux of antibiotics across explants were measured. DON increased the permeability of small intestine explants, expressed by a reduction in TEER and an intensification of transcellular marker transport. DON did not affect AMX transport, but it accelerated DOX transport by approximately five times. The results suggest that DON inhibits the efflux transport of DOX to the intestinal lumen, and thus significantly changes its absorption from the gastrointestinal tract.

Abstract 374: Small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs

Abstract Although oral dosing is the most convenient route of drug administration, most anti-neoplastic drugs are administered intravenously due to poor bioavailability because of the epithelial barriers. A major organ involved in oral drug absorption is the small intestine where enterocytes are linked together by tight junctions (TJs). TJs regulate paracellular transport by creating a selectively-permeable barrier to small molecules, ions and water, thereby restricting the uptake of drugs. Transient modulation of the TJs is considered a potential strategy to improve drug delivery. Human small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. However, their geometry prevents access to the apical compartment of the epithelium (the outer, or luminal, surface being located within the organoid with the basal surface on the outside), making them unsuitable for the study of orally-administered drugs. Here we describe methods to generate transwell cell monolayers and inverted or ‘apical-out’ organoids, both derived from normal human small intestine, that allow direct access to the apical surface. We demonstrate that both formats provide an in vivo-like cell composition, comprising of all the intestinal cellular types, including stem cells, Paneth cells, goblet cells, enteroendocrine cells and enterocytes, and respond to treatment with ‘tool drug’ TJ modulators such as sodium caprate, chitosan, EGTA and various cytokines. Briefly, for the generation of transwell-grown cell monolayers, SI organoids were dissociated into single cells and plated in matrix-coated transwell inserts. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements. Treatments with TJ modulators were applied after TEER reached a plateau and effects on TJs were evaluated by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. For the generation of apical-out organoids, SI organoids were mechanically broken into fragments and seeded in low adhesion plates until the organoids switched polarity. Treatments with TJ modulators were applied and effects on TJs were evaluated by immunofluorescence. In conclusion, our models provide an innovative in vitro solution to study the effect of TJ modulators on increasing paracellular transport of orally-administered, anti-neoplastic drugs. Furthermore, as anti-neoplastic drugs can also have toxic effects on the intestinal epithelium by affecting TJs, thereby creating a leaky barrier, the models are useful to predict such effects and stratify drug candidates prior to in vivo studies. Citation Format: Valentina Ubertini, Stephanie French, James Wilson, Frida Ponthan. Small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 374.

Preclinical In Vitro Model to Assess the Changes in Permeability and Cytotoxicity of Polarized Intestinal Epithelial Cells during Exposure Mimicking Oral or Intravenous Routes: An Example of Arsenite Exposure.

The gastrointestinal tract (GIT) is exposed to xenobiotics, including drugs, through both: local (oral) and systemic routes. Despite the advances in drug discovery and in vitro pre-clinical models, there is a lack of appropriate translational models to distinguish the impact of these routes of exposure. Changes in intestinal permeability has been observed in different gastrointestinal and systemic diseases. This study utilized one such xenobiotic, arsenic, to which more than 200 million people around the globe are exposed via their food, drinking water, work environment, soil, and air. The purpose of this study was to establish an in vitro model to mimic gastrointestinal tract exposure to xenobiotics via oral or intravenous routes. To achieve this, we compared the route (mimicking oral and intravenous exposure to GIT and the dose response (using threshold approach) of trivalent and pentavalent inorganic arsenic species on the permeability of in vitro cultured polarized T84 cells, an example of intestinal epithelial cells. Arsenic treatment to polarized T84 cells via the apical and basolateral compartment of the trans-well system reflected oral or intravenous routes of exposure in vivo, respectively. Sodium arsenite, sodium arsenate, dimethyl arsenic acid sodium salt (DMAV), and disodium methyl arsonate hydrate (MMAV) were assessed for their effects on intestinal permeability by measuring the change in trans-epithelial electrical resistance (TEER) of T-84 cells. Polarized T-84 cells exposed to 12.8 µM of sodium arsenite from the basolateral side showed a marked reduction in TEER. Cytotoxicity of sodium arsenite, as measured by release of lactate dehydrogenase (LDH), was increased when cells were exposed via the basolateral side. The mRNA expression of genes related to cell junctions in T-84 cells was analyzed after exposure with sodium arsenite for 72 h. Changes in TEER correlated with mRNA expression of focal-adhesion-, tight-junction- and gap-junction-related genes (upregulation of Jam2, Itgb3 and Notch4 genes and downregulation of Cldn2, Cldn3, Gjb1, and Gjb2). Overall, exposure to sodium arsenite from the basolateral side was found to have a differential effect on monolayer permeability and on cell-junction-related genes as compared to apical exposure. Most importantly, this study established a preclinical human-relevant in vitro translational model to assess the changes in permeability and cytotoxicity during exposure, mimicking oral or intravenous routes.

Inflammation of Gill Epithelia in Fish Causes Increased Permeation of Petrogenic Polar Organic Chemicals via Disruption of Tight Junctions.

The epithelial cell layer that lines the gills of fish controls paracellular permeation of chemicals through tight junctions. The integrity of tight junctions can be affected by inflammation, which likely affects the bioavailability of chemicals. Here, the inflammation of the rainbow trout gill cell line RTgill-W1 was induced via exposure to bacterial lipopolysaccharides (LPS). Cells were then coexposed to extracts of oil sands process-affected water (OSPW), which contain complex mixtures of chemicals. After 24 h of exposure, cells exposed to LPS showed a reduction in transepithelial electrical resistance, an indicator of tight junction integrity. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis determined that abundances of transcripts of genes coding for tight junction proteins were significantly less in cells exposed to 20, 50, or 100 mg L-1 LPS. Chemical analysis revealed increased permeation of constituents of OSPW across epithelia at all studied LPS concentrations. These in vitro findings were confirmed in vivo in rainbow trout exposed to LPS and OSPW for 48 h, which resulted in greater accumulation of chemicals relative to that for fish exposed to OSPW alone. Our results demonstrated that inflammation and disruption of tight junctions could lead to greater uptake of potentially harmful chemicals from the environment, which has implications for mixture risk assessment.

Xuebijing Injection Ameliorates H2S-Induced Acute Respiratory Distress Syndrome by Promoting Claudin-5 Expression.

To investigate the protective effects and underlying mechanisms of Xuebijing Injection (XBJ) on the lung endothelial barrier in hydrogen sulfide (H2S)-induced acute respiratory distress syndrome (ARDS). Sprague-Dawley rats were exposed to H2S (300 ppm) to establish ARDS model, while human pulmonary microvascular endothelial cells (HPMECs) were incubated with NaHS (a H2S donor, 500 µmol/L) to establish cell model. H2S and XBJ were concurrently administered to the rat and cell models. Lung hematoxylin and eosin staining, immunohistochemistry, transmission electron microscopy and wet/dry ratio measurement were used to confirm ARDS induced by H2S in vivo. The expression levels of claudin-5, phosphorylated protein kinase B (p-AKT)/t-AKT and p-forkhead box transcription factor O1 (FoxO1)/t-FoxO1 in vivo and in vitro were also assessed. Paracellular permeability and transepithelial electrical resistance (TEER) were measured to evaluate endothelial barrier function in the cell model. The morphological investigation showed that XBJ attenuated H2S-induced ARDS in rats. XBJ significantly ameliorated both the reduction in TEER and the increased paracellular permeability observed in NaHS-treated HPMECs (P<0.05). The protective effects of XBJ were blocked by LY294002, a phosphatidylinositol 3-kinase (PI3K)/AKT/FoxO1 pathway antagonist (P<0.05). Furthermore, XBJ promoted the expression of claudin-5 and increased the levels of p-AKT and p-FoxO1 in vivo and in vitro (P<0.05). XBJ ameliorated H2S-induced ARDS by promoting claudin-5 expression via the PI3K/AKT/FoxO1 signaling pathway.

Pharmacokinetic interactions: The effects of selected herbal extracts on permeation of P-glycoprotein substrate drugs across excised pig intestinal tissue

Introduction: There is growing concern that co-administered herbal medicines may alter the pharmacokinetics and, therefore, the efficacy and toxicity of Western drugs. The aim of this study was to investigate the potential membrane permeation modulating effects of four herbal extracts, i.e., Harpagophytum procumbens, Hoodia gordonii, Leonotis leonurus, and Vitis vinifera on a model compound, Rhodamine 123 (RH-123). Methods: An in vitro permeation model, i.e., excised pig intestinal tissue, mounted to test chambers in a Sweetana-Grass diffusion apparatus, was used to measure the bi-directional transport of RH-123 in the presence and in the absence of four herbal extracts. The concentration of transported RH-123 in each sample was determined by means of fluorescence spectroscopic analysis. The integrity of the mounted jejunum tissue during experimentation was confirmed by measuring the permeation of Lucifer Yellow through these membranes. Trans-epithelial electrical resistance (TEER) of the mounted membranes was also measured at the onset and termination of each experiment to monitor whether tight junction modulation occurred. Results: H. procumbens extract increased the secretory transport of RH-123, indicative of the induction of P-glycoprotein (P-gp) mediated efflux. H. gordonii extract also increased RH-123’s absorptive transport, coupled with a subsequent decrease in its secretory transport, indicating the P-gp related efflux inhibition. Contrary, L. leonurus extract reduced RH-123’s absorptive transport, accompanied by an increase in its secretory transport. V. vinifera seed extract, however, increased both the absorptive and secretory transport of RH-123. A reduction in TEER was observed in the presence of V. vinifera extract, indicating the modulation of tight junction integrity. Conclusion: The ex vivo pharmacokinetics interactions recorded in the current study suggest that the co-administration of herbal medicines could alter the extent of membrane permeation of Western drugs.

Beneficial Effects of Quercetin on Microcystin-LR Induced Tight Junction Defects.

Quercetin has numerous functions including antioxidant and anti-inflammatory effects. The beneficial effect of quercetin against microcystin-LR (MC-LR)-induced testicular tight junctions (TJs) defects in vitro and in vivo were investigated. Significant reductions in transepithelial electrical resistance, occludin, and zonula occludens-1(ZO-1) levels were detected in the MC-LR-treated TM4 cells, and quercetin attenuated these effects. Interestingly, quercetin suppressed MC-LR-induced phosphorylation of protein kinase B (AKT). It effectively inhibited the accumulation of reactive oxygen species (ROS) in cells stimulated by MC-LR. In addition, ROS inhibitors blocked the TJ damage that is dependent on the AKT signaling pathway induced by MC-LR. In conclusion, our results suggest that alleviates MC-LR-impaired TJs by suppressing the ROS-regulated activation of the AKT pathway.

PGC-1\u03b1 regulates airway epithelial barrier dysfunction induced by house dust mite

BackgroundThe airway epithelial barrier function is disrupted in the airways of asthmatic patients. Abnormal mitochondrial biogenesis is reportedly involved in the pathogenesis of asthma. However, the role of mitochondrial biogenesis in the airway barrier dysfunction has not been elucidated yet. This study aimed to clarify whether the peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), a central regulator of mitochondrial biogenesis, is involved in the disruption of the airway barrier function induced by aeroallergens.MethodsBEAS-2B cells were exposed to house dust mite (HDM) and the expressions of PGC-1α and E-cadherin, a junctional protein, were examined by immunoblotting. The effect of SRT1720, a PGC-1α activator, was investigated by immunoblotting, immunocytochemistry, and measuring the transepithelial electrical resistance (TEER) on the HDM-induced reduction in mitochondrial biogenesis markers and junctional proteins in airway bronchial epithelial cells. Furthermore,the effects of protease activated receptor 2 (PAR2) inhibitor, GB83, Toll-like receptor 4 (TLR4) inhibitor, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), protease inhibitors including E64 and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) on the HDM-induced barrier dysfunction were investigated.ResultsThe amounts of PGC-1α and E-cadherin in the HDM-treated cells were significantly decreased compared to the vehicle-treated cells. SRT1720 restored the expressions of PGC-1α and E-cadherin reduced by HDM in BEAS-2B cells. Treatment with SRT1720 also significantly ameliorated the HDM-induced reduction in TEER. In addition, GB83, LPS-RS, E64 and AEBSF prevented the HDM-induced reduction in the expression of PGC1α and E-cadherin.ConclusionsThe current study demonstrated that HDM disrupted the airway barrier function through the PAR2/TLR4/PGC-1α-dependent pathway. The modulation of this pathway could be a new approach for the treatment of asthma.

Induction of permeability and apoptosis in colon cancer cell line with chitosan

The effect of chitosan on tight junctions (TJs) permeability on Caco-2 cell monolayer intestinal model was investigated. We have also investigated the effects of low molecular weight water-soluble chitosan (LMWC) on apoptosis in HT-29 cell. The changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance (TEER) and permeability to Lucifer yellow, were assessed in response to chitosan treatment. High molecular weight chitosan (HMWC) was found to cause up to 50% dose-dependent reduction in TEER of Caco-2 cell monolayer without damage to the cell membrane under lower concentration. The effect of HMWC on TJs was confirmed by increased permeability of lucifer yellow when cells were treated with 0.001-0.0001% HMWC for 120 min compared to control cells. Results showed that HMWC did not affect the F-actin of cytoskeleton. LMWC was proven to be an antitumor compound as shown by inducing apoptosis as a function of DNA fragmentation. These results suggest that HMWC is an useful drug delivery agent in paracellular pathway and LMWC has potential value in colon cancer therapy.