Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability

Background: Small intestine permeability is frequently altered in inflammat ory bowel disease and may be caused by the translocation of intestinal toxi ns-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 . Materials and Methods: In vitro study, MDCK (Madin-Darby Canine Kidney) cel ls were exposed to H2O2 (100 mu M for 2h), or zymosan (200 mu l of stock:so lution 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 w ith n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mi ce lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) w ere treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). I n 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. Results: Exposure of MDCK cells to hydrogen peroxide caused a significant i mpairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A si gnificant increase of nitrate and nitrite levels, stable metabolites of nit ric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence a nd impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junction al damage as well as reduced the NO-induced occludin damage. In addition, H 2O2 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 permea bility to lanthanum nitrate (molecular weight, 433) in the terminal ileal T Js in zymosan-treated iNOSWT mice compared with permeableTJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or s trand depth in the ilea from control or treated animals. In addition, a sig nificant disrupted immunofluorescence signal for occludin, ZO-1 and beta-ca tenin was observed in the terminal ilea of zymosan-treated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular d istribution patterns of occludin, ZO-1 and beta-catenin. Similarly NAC or 3 -AB treatments were able to prevent zymosan-induced damage of junctional pr oteins in iNOSWT mice. Conclusion: In conclusion, this study demonstrates that the alteration of p ermselectivity is most likely induced by ROS and PARS activation.