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Abstract
Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with permeable TJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or strand depth in the ilea from control or treated animals. In addition, a significant disrupted immunofluorescence signal for occludin, ZO-1 and beta-catenin was observed in the terminal ilea of zymosan-treated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular distribution patterns of occludin, ZO-1 and beta-catenin. Similarly NAC or 3-AB treatments were able to prevent zymosan-induced damage of junctional proteins in iNOSWT mice. In conclusion, this study demonstrates that the alteration of permselectivity is most likely induced by ROS and PARS activation.
Highlights
The mucosa of the digestive tract provides an effective barrier to the entry of intestinal bacte-ria and endotoxins in healthy individuals
We found an increase of tight junctional permeability to lanthanum nitrate in the terminal ileal tight junctions (TJ) in zymosan-treated iNOSWT mice compared with permeableTJ in the control animals
Exposure of Madin-Darby Canine Kidney (MDCK) to H2O2 (100 M) for 2 hr caused a significant impairment of mitochondrial respiration (Fig. 1B) that was associated with a reduction of cell adherence, as well as derangement of the occludin protein
Summary
The mucosa of the digestive tract provides an effective barrier to the entry of intestinal bacte-ria and endotoxins in healthy individuals. As has been shown in animal studies [3,4], this barrier function can be lost during critical illness and, may lead to translocation of bacteria and absorption of endotoxins into the circulation. Cuzzocrea et al.: Free Radicals and TJ Permeability 767 translocation of bacteria and absorption of endotoxins may have profound systemic effects and may result in bacteremia, as well as endotoxemia. Endotoxins, in turn, increase the permeability of the digestive tract, thereby facilitating bacterial translocation and endotoxin absorption [3,4]. The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. Results: Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after
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