Thalidomide treatment reduces the alteration of paracellular barrier function in mice ileum during experimental colitis.

Small intestine permeability is frequently altered in inflammatory bowel diseases and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJs) and adherence. Thus, the aim of the present study was to examine the effects of thalidomide treatment on the permeability and structure of small intestine TJs in an animal model of experimental colitis induced by dinitrobenzene sulfonic acid (DNBS). Four days after colitis induction with DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin and zonula occludens 1. When compared with DNBS-treated mice, thalidomide-treated (200 mg/kg orally starting 30 min after the administration of DNBS) mice subjected to DNBS-induced colitis experienced a significantly reduced rate of the extent and severity of the histological signs of colon injury associated with a significant reduction of plasma and colon tumor necrosis factor alpha levels. After administration of DNBS to the mice induced a significant increase of ileal permeability was observed. Distal colitis in mice induced an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. In particular, we have observed that thalidomide treatment resulted in a significant reduction of the following: (1) the degree of colon injury, (2) the alteration of zonula occludens 1 and occludin localization (immunohistochemistry), and (3) intestinal permeability caused by DNBS in the colon. Taken together, our results clearly show that thalidomide treatment reduced small intestinal permeability in experimental colitis through the regulation of TJ protein.

Mazzon E ，Cuzzocrea S 《SHOCK》

5-lipoxygenase modulates the alteration of paracellular barrier function in mice ileum during experimental colitis.

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 (TJs) and adherence. Recently, it has been shown that 5-lipoxygenase (5-LO) plays an important role in the development of various inflammatory conditions like inflammatory bowel disease. In the present study, by comparing the responses in wild-type mice (5-LOWT) with those of mice lacking the 5-lipoxygenase (5-LOKO), we investigated the role played by this enzyme in the permeability and structure of small intestine TJs in an animal model of experimental colitis. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). Four days after colitis induction by DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin and ZO-1. When compared with DNBS-treated 5-LOWT mice, DNBS-treated 5-LOKO mice experienced a reduced rate of the extent and severity of the histological signs of colon injury. After administration of DNBS, 5-LOWT mice showed a significant increase of ileal permeability (88.3% +/- 1.2%) compared with sham (5.6% +/- 0.5%). In colitis, the percentage of "leaky" junctions in terminal ilea correlated positively with the macroscopic colon damage score. Distal colitis in 5-LOWT mice induces an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. On the contrary, a significant reduction of (1) the degree of colon injury, (2) the alteration of ZO-1 and occludin localization (immunohistochemistry), and (3) ileal permeability (8.1% +/- 0.7%) caused by DNBS in the colon was observed in 5-LOKO mice. Similarly, the treatment of 5-LOWT with zileuton (50 mg/kg per oral gavage twice a day), a 5-LO inhibitor, resulted in a significant reduction of all the previously described parameters. Taken together, our results clearly demonstrate that 5-LO modulates small intestinal permeability in experimental colitis through the regulation of TJ protein.

Mazzon E ，Sautebin L ，Caputi AP ，Cuzzocrea S ... -  《SHOCK》

Absence of functional peroxisome proliferator-activated receptor-alpha enhanced ileum permeability during experimental colitis.

The aim of the present study was to examine the role of endogenous peroxisome proliferator-activated receptor-alpha (PPAR-alpha) ligand on the permeability and structure of small intestine tight junctions (TJs) in an animal model of experimental colitis, induced by dinitrobenzene sulfuric acid (DNBS). Four days after colitis induction with DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin, zonula occludens 1, and claudin 2. Administration of DNBS to wild-type mice induced colon injury associated with a significant increase of plasma and colon tumor necrosis factor-alpha levels and with a significant increase of ileal permeability. Distal colitis in mice induced an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. Small intestinal permeability was associated with the presence of apoptosis (evaluated by FAS ligand expression and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling coloration), which was associated with a significantly increased expression of proapoptotic Bax and decreased ileum content of antiapoptotic Bcl-2. Absence of a functional PPAR-alpha gene in PPAR-alpha knockout mice resulted in a significant augmentation of all the above-described parameters. Taken together, our results clearly demonstrate that endogenous PPAR-alpha ligands reduced small intestinal permeability in experimental colitis through the regulation of apoptosis and TJ protein.

Mazzon E ，Cuzzocrea S 《SHOCK》

Thalidomide treatment reduces colon injury induced by experimental colitis.

The immunological and genetic pathogeneses of inflammatory bowel disease (IBD) have been well studied but not well elucidated in the recent years. Accordingly, the pharmacological treatment of IBDs is focusing upon the individual pathologic step (targeting therapy). It has been shown recently that new drugs such as biological immunomodulating agents and anti-inflammatory cytokines have better short-term effects in some respects than the conventional drugs, and they might change the treatment strategy of IBDs in the near future. The aim of the present study was to examine the effects of thalidomide treatment in the development of experimental colitis. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). DNBS-treated mice experienced diarrhea and weight loss. At 4 days after administration of DNBS, the mucosa of the colon exhibited large areas of necrosis. The observed mucosa alteration was associated with the colon production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and vascular endothelial growth factor (VEGF). Neutrophil infiltration (determined by histology as well as an increase in myeloperoxidase activity in the mucosa) was associated with an upregulation of intercellular adhesion molecule-1. Immunohistochemistry for nitrotyrosine and poly (ADP ribose) showed an intense staining in the inflamed colon. When compared with DNBS-treated mice, thalidomide-treated (200 mg/kg orally) mice subjected to DNBS-induced colitis experienced a significantly lower rate in the extent and severity of the histological signs of colon injury. Thalidomide also caused a substantial reduction of the rise in myeloperoxidase activity (mucosa), in the increase in the tissue levels of TNF-alpha, IL-1beta, and VEGF, in the increase in staining (immunohistochemistry) for nitrotyrosine and for poly (ADP ribose), as well as in the upregulation of intercellular adhesion molecule-1 caused by DNBS in the colon. Thus, thalidomide treatment reduces the degree of colitis caused by DNBS. We propose that this evidence may help to clarify the therapeutic actions of thalidomide in patients with Crohn's disease.

Mazzon E ，Muià C ，Di Paola R ，Genovese T ，De Sarro A ，Cuzzocrea S ... -  《SHOCK》

Role of iNOS in hepatocyte tight junction alteration in mouse model of experimental colitis.

A variety of hepatobiliary abnormalities occur in inflammatory bowel diseases (IBDs). The role of tight junction (TJ) in hepatobiliary complications have been well described. The purpose of this study was to investigate the role of inducible nitric oxide (NOS) in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated iNOS wild-type (WT) mice, DNBS-treated iNOS knock out mice (iNOSKO) mice experienced a significant less rate of the extent and severity of the histological signs of colon injury. Colon levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also significantly reduced in iNOS-KO mice in comparison to wild-type mice. Liver histology from iNOSKO and wild-type mice iNOSWT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and alanine aminotransferase, were significantly reduced in DNBS-iNOSKO mice vs DNBS-iNOSKO mice. Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice, lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional iNOS gene in iNOSKO mice resulted in a significant reduction of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labeling of frozen liver sections from DNBS-iNOSWT mice showed a significant alteration of the immunolocalization for claudin-1 and zonula occludens (ZO)-1. In contrast, a significant reduced alteration in the localization of the immunosignals for claudin-1 and ZO-1 was observed in the liver from iNOSKO mice after DNBS administration. In conclusion, we suggest that the iNOS may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.

Mazzon E ，Cuzzocrea S 《CELLULAR AND MOLECULAR BIOLOGY》