Celecoxib, a selective cyclo-oxygenase-2 inhibitor reduces the severity of experimental colitis induced by dinitrobenzene sulfonic acid in rats

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Abstract

Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leukocyte infiltration, upregulation of the expression of intercellular adhesion molecule 1 (ICAM-1) and upregulation of P-selectin in the colon. Here, we investigate the effects of the selective cyclo-oxygenase-2 inhibitor, celecoxib, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Rats experienced hemorrhagic diarrhoea and weight loss. At 4 days after administration of DNBS, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology, as well as an increase in myeloperoxidase activity in the mucosa) was associated with upregulation of ICAM-1 and P-selectin, as well as high tissue levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) polymerase showed intense staining in the inflamed colon. Celecoxib (5 mg/kg twice a day orally) significantly reduced the degree of hemorrhagic diarrhoea and the weight loss caused by administration of DNBS. Celecoxib also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde, (iv) the increase in staining (immunohistochemistry) for nitrotyrosine, as well as (v) the upregulation of ICAM-1 and P-selectin caused by DNBS in the colon. Thus, we provide the first evidence that a selective cyclo-oxygenase-2 inhibitor celecoxib reduces the degree of colitis caused by DNBS.

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit prostanoid generation and their effects vary in different tissues and organs. In the upper gastrointestinal tract, prostanoids are essential for the maintenance of mucosal integrity and, therefore, inhibition of their generation induced by NSAIDs results in gastrointestinal bleeding and ulceration (Whittle et al., 1986). The function of the two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2 in maintaining mucosal homeostasis and modulating inflammation in the digestive tract remains uncertain. Colonic prostanoid generation is increased in both experimental colitis and in inflammatory bowel disease Sharon et al., 1978, Rachmilewitz et al., 1989 and correlates well with disease activity Sharon et al., 1978, Rachmilewitz et al., 1989, Allgayer et al., 1989. Therefore, prostaglandins also play a protective role against gastrointestinal injury (Hoult and Moore, 1978) and downregulation of the expression of proinflammatory cytokines Knudsen et al., 1986, Marcinkiewicz, 1991. Indeed, experimental colitis can be attenuated by pretreatment with exogenous prostaglandins Allgayer et al., 1989, Fedorak et al., 1990, and prostaglandin E2 plays a major role in the regeneration of the epithelial crypts after dextran sodium sulfate (DSS)- and radiation-induced intestinal damage in mice (Cohn et al., 1997). Furthermore, inhibition of prostaglandin synthesis by indomethacin induces acute and chronic enterocolitis in genetically susceptible rats (Yamada et al., 1993), which is consistent with the gastrointestinal ulceration seen in patients treated with nonsteroidal anti-inflammatory drugs (NSAIDs) Kaufmann and Taubin, 1987, Bjarnson et al., 1993. This effect is attributed to the inhibition of constitutive mucosal prostaglandins, which have cytoprotective properties (Wallace et al., 1992). The relative contribution of cyclooxygenase-1 and cyclooxygenase-2 isoforms to the biological actions of prostaglandins in the gastrointestinal mucosa is less clear. Several observations led to the hypothesis that inducible cyclooxygenase-2 drives the proinflammatory actions of prostaglandins during mucosal injury, whereas cyclooxygenase-1 regulates gastrointestinal homeostasis through the synthesis of cytoprotective prostaglandins (Masferrer et al., 1996). In mice, cyclooxygenase-1 has been reported to play a protective role against small intestine (Cohn et al., 1997) and colon (Tessner et al., 1998) mucosal injury through the synthesis of prostaglandins that promote epithelial regeneration. In contrast, cyclooxygenase-2 expression is induced during inflammation (Seibert et al., 1994), and its expression is inhibited by endogenous glucocorticoids (O'Banion et al., 1991). Based on these observations, some researchers have attributed the anti-inflammatory action of NSAIDs to the inhibition of cyclooxygenase-2, and the harmful effects of NSAIDs on the gastrointestinal mucosa are attributed to the blockade of cyclooxygenase-1 activity Xie et al., 1992, Chan et al., 1995. This association of cyclooxygenase-2 with inflammatory events led to the development of selective cyclooxygenase-2 inhibitors expected to display systemic anti-inflammatory properties, while avoiding gastrointestinal toxicity. Recently, a clinical trial in patients with osteoarthritis emphasized the safety of the selective cyclooxygenase-2 inhibitor celecoxib, which caused significantly less gastroduodenal ulceration than did the NSAIDs (Goldstein et al., 2000). However, cyclooxygenase-2 inhibitors have also been shown to be harmful when there is preexisting gastrointestinal inflammation because they delay gastric ulcer healing in mice (Mizuno et al., 1997) and exacerbate colon mucosal inflammation in rats (Reuter et al., 1996). In addition, it has been recently demonstrated that some cyclooxygenase-2 inhibitor did not exerts any beneficial effect in an experimental model of colitis induced by tinitrobenzene sulfonic acid (TNBS) (Lesch et al., 1999) and that cyclooxygenase-2 derived prostaglandin D2 is an early anti-inflammatory signal in experimental colitis.

The role of NSAIDs in the possible modulation of colon inflammation is controversial and remain uncertain.

In this study, we have investigated the effect of celecoxib, a new cyclooxygenase-2 selective inhibitor, on the inflammatory response (colitis) caused by intracolonic administration of DNBS. In particular, we investigated the effects of celecoxib on the colon injury associated with DNBS-induced colitis. In order to gain a better insight into the mechanism of action of celecoxib, we have determined the following endpoints of the inflammatory response: (1) polymorphonucleates infiltration, (2) peroxynitrite formation (immunohistochemistry), (3) activation of the nuclear enzyme poly(ADP-ribose) polymerase, (4) expression of cyclooxygenase-2 protein (immunohistochemistry) and activity, (5) lipid peroxidation and (6) colon injury.

Section snippets

Animals

Male Sprague–Dawley rats (300–350 g; Charles River, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (D.M. 116192), as well as with EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

Experimental groups

Celecoxib was given twice a day as an oral bolus (5 mg/kg, DNBS+celecoxib group; n=10), the first dose was given immediately

Effects of celecoxib on the degree of colitis (histology)

Four days after intracolonic administration of DNBS, the colon appeared flaccid and filled with liquid stool. The cecum, colon and rectum showed evidence of mucosal congestion, erosion and hemorrhagic ulcerations (see Fig. 1 for damage score). The histopathological features included necrosis and edema and diffuse inflammatory cells infiltration in the mucosa (Fig. 2B). The inflammatory changes of the intestinal tract were associated with an increase in the weight of the colon (Fig. 3B).

Discussion

Our data show that rats treated with celecoxib, a new cyclooxygenase-2 selective inhibitor, are significantly more resistant to lethality and pathological changes in the colon and rectum associated with DNBS-induced colitis.

Prostanoid generation was found to be enhanced in the colonic mucosa of patients with ulcerative colitis and Crohn's disease (Sharon et al., 1978). In view of the role of prostanoids as mediators of symptoms associated with inflammation, trials to modulate inflammatory bowel

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