Homoectoine Protects Against Colitis by Preventing a Claudin Switch in Epithelial Tight Junctions.

BACKGROUND: Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins. AIM: To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice. METHODS/RESULTS: We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis. CONCLUSION: While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.

Therapeutic effects of different doses of polyethylene glycosylated porcine glucagon-like peptide-2 on ulcerative colitis in male rats.

BACKGROUND: Polyethylene glycosylated (PEGylated) porcine glucagon-like peptide-2 (pGLP-2) considerably increases half-life and stability compared with the native pGLP-2, but the effective dose for intestinal damage is still unclear. This study aims to evaluate the available dose of polyethylene glycosylated porcine glucagon-like peptide-2 (PEG-pGLP-2), a modified, long-acting form of pGLP-2 in an experimental rat model of ulcerative colitis. METHODS: Thirty-five male rats were randomly assigned into five groups: control, dextran sodium sulphate (DSS), DSS + PEG-pGLP-2(L), DSS + PEG-pGLP-2(M) and DSS + PEG-pGLP-2(H). Rats in control group received only water; other rats were fed with 5% (w/v) DSS and intraperitoneally administered with 12.5, 25 and 100 nmol/kg PEG-pGLP-2 daily for 6 days. RESULTS: Compared with the control treatment, DSS treatment significantly (p < 0.05) decreased body weight change, colonic length, duodenal villus height and expression of zonula occludens-1, whereas significantly (p < 0.05) increased colonic damage score and expression of claudin-1, interleukin (IL)-1, IL-7, IL-10, interferon-γ and tumour necrosis factor (TNF)-α in colon. However, the three doses of PEG-pGLP-2 all reduced these effects; these treatments significantly (p < 0.05) increased body weight change and duodenal villus height, whereas significantly (p < 0.05) decreased colonic damage score and expression of IL-1, IL-7 and TNF-α in colon. Specifically, low-dose (12.5 nmol/kg/d) PEG-pGLP-2 was effective. CONCLUSIONS: These results indicated that PEG-pGLP-2 is a novel and potentially effective therapy for intestinal healing in a relatively low dose.

Chloroquine and Hydroxychloroquine Increase Retinal Pigment Epithelial Layer Permeability.

Antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used as antiinflammatory drugs, but side effects include retinopathy and vision loss. The objective of this study was to examine the effect of CQ and HCQ on the barrier integrity of retinal pigment epithelial (RPE) cell monolayers in vitro. Permeability of ARPE-19 cell monolayers was determined using Fluorescein isothiocyanate (FITC)-labeled dextran. The influence of CQ and HCQ on cell death and the expression tight junction molecules was examined. CQ and HCQ significantly increased ARPE-19 monolayer permeability after 3 and 18 h, respectively, and enhanced mRNA levels for claudin-1 and occludin. Cytotoxicity was only observed after 18 h exposure. Thus, CQ and HCQ rapidly enhance RPE barrier permeability in vitro, independent of cytotoxicity or loss of zonula occludens-1, claudin-1, and occludin expression. Our findings suggest that CQ/HCQ-induced permeability of the RPE layer may contribute to blood-retinal barrier breakdown in case of CQ/HCQ-induced retinopathy.

Oral activated charcoal adsorbent (AST-120) ameliorates chronic kidney disease-induced intestinal epithelial barrier disruption.

BACKGROUND: Chronic kidney disease (CKD) impairs intestinal barrier function which by allowing influx of noxious products causes systemic inflammation. We have recently shown that intestinal barrier dysfunction in CKD is due to degradation of epithelial tight junction (TJ) which is, in part, mediated by influx of urea and its conversion to ammonia by microbial urease. We hypothesized that by adsorbing urea and urea-derived ammonia, oral activated charcoal (AST-120) may ameliorate CKD-induced intestinal epithelial barrier disruption and systemic inflammation. METHODS: Rats were randomized to the CKD or control groups. The CKD group was fed a chow containing 0.7% adenine for 2 weeks. They were then randomized to receive a chow with or without AST-120 (4 g/kg/day) for 2 weeks. Rats consuming regular diet served as controls. Animals were then euthanized, colons were removed and processed for Western blot and immunohistology, and plasma was used to measure endotoxin and oxidative and inflammatory markers. RESULTS: Compared with the controls, the untreated CKD rats showed elevated plasma endotoxin, IL-6, TNF-α, MCP-1, CINC-3, L-selectin, ICAM-1, and malondialdehyde, and depletions of colonic epithelial TJ proteins, claudin-1, occludin, and ZO1. Administration of AST-120 resulted in partial restoration of the epithelial TJ proteins and reduction in plasma endotoxin and markers of oxidative stress and inflammation. CONCLUSIONS: CKD animals exhibited depletion of the key protein constituents of the colonic epithelial TJ which was associated with systemic inflammation, oxidative stress and endotoxemia. Administration of AST-120 attenuated uremia-induced disruption of colonic epithelial TJ and the associated endotoxemia, oxidative stress and inflammation.

Protective effects of N-acetylcysteine on acetic acid-induced colitis in a porcine model.

BACKGROUND: Ulcerative colitis is a chronic inflammatory disease and involves multiple etiological factors. Acetic acid (AA)-induced colitis is a reproducible and simple model, sharing many characteristics with human colitis. N-acetylcysteine (NAC) has been widely used as an antioxidant in vivo and in vitro. NAC can affect several signaling pathways involving in apoptosis, angiogenesis, cell growth and arrest, redox-regulated gene expression, and inflammatory response. Therefore, NAC may not only protect against the direct injurious effects of oxidants, but also beneficially alter inflammatory events in colitis. This study was conducted to investigate whether NAC could alleviate the AA-induced colitis in a porcine model. METHODS: Weaned piglets were used to investigate the effects of NAC on AA-induced colitis. Severity of colitis was evaluated by colon histomorphology measurements, histopathology scores, tissue myeloperoxidase activity, as well as concentrations of malondialdehyde and pro-inflammatory mediators in the plasma and colon. The protective role of NAC was assessed by measurements of antioxidant status, growth modulator, cell apoptosis, and tight junction proteins. Abundances of caspase-3 and claudin-1 proteins in colonic mucosae were determined by the Western blot method. Epidermal growth factor receptor, amphiregulin, tumor necrosis factor-alpha (TNF-α), and toll-like receptor 4 (TLR4) mRNA levels in colonic mucosae were quantified using the real-time fluorescent quantitative PCR. RESULTS: Compared with the control group, AA treatment increased (P < 0.05) the histopathology scores, intraepithelial lymphocyte (IEL) numbers and density in the colon, myeloperoxidase activity, the concentrations of malondialdehyde and pro-inflammatory mediators in the plasma and colon, while reducing (P < 0.05) goblet cell numbers and the protein/DNA ratio in the colonic mucosa. These adverse effects of AA were partially ameliorated (P < 0.05) by dietary supplementation with NAC. In addition, NAC prevented the AA-induced increase in caspase-3 protein, while stimulating claudin-1 protein expression in the colonic mucosa. Moreover, NAC enhanced mRNA levels for epidermal growth factor and amphiregulin in the colonic mucosa. CONCLUSION: Dietary supplementation with NAC can alleviate AA-induced colitis in a porcine model through regulating anti-oxidative responses, cell apoptosis, and EGF gene expression.

Entamoeba histolytica EhCP112 Dislocates and Degrades Claudin-1 and Claudin-2 at Tight Junctions of the Intestinal Epithelium.

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.

Cell junction remodeling in gingival tissue exposed to a microbial toxin.

UNLABELLED: The gingival epithelium plays a key role in protecting the supporting structures of the teeth from bacteria and their products. In ex vivo experiments, we recently showed that the cytolethal distending toxin (Cdt) from the periodontal pathogen Aggregatibacter actinomycetemcomitans causes extensive damage to gingival tissue. Morphological changes included detachment of the keratinized outer layer, distention of spinous and basal cells in the oral epithelium, disruption of rete pegs, and apparent dissolution of cell junctions. Adherens junctions (zonula adherens) are essential for maintaining barrier function and integrity of gingival epithelium. Therefore, immunohistochemical and RT-PCR analyses of human gingival explants (HGX) and human gingival epithelial cells (HGEC) were utilized for a closer examination of the effects of the Cdt on E-cadherin, the key membrane component of adherens junctions. Although there was some variability among tissue donors, exposure of gingival tissue or isolated epithelial cells to the toxin generally resulted in a pronounced increase in the expression and cytosolic distribution of E-cadherin, accompanied by an increase in levels of the intracellular scaffolding proteins ß-catenin and ß-actin. These results indicate that the Cdt induced substantial remodeling of adherens junctions, with a potential impact on the barrier function of gingival epithelium. ABBREVIATIONS: cytolethal distending toxin (Cdt), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), transepithelial resistance (TER).