Rosiglitazone, a PPARγ ligand, modulates signal transduction pathways during the development of acute TNBS-induced colitis in rats

https://doi.org/10.1016/j.ejphar.2007.01.047Get rights and content

Abstract

Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARγ), a highly nuclear receptor expressed in the colon, may participate in the control of inflammation, especially in regulating the production of immunomodulatory and inflammatory mediators, cellular proliferation and apoptosis. In order to delve into the anti-inflammatory mechanisms and signalling pathways of PPARγ agonists, we have studied the effects of rosiglitazone, a PPARγ agonist on the extent and severity of acute ulcerative colitis caused by intracolonic administration of 2,4,6-trinitribenzene sulfonic acid (TNBS) in rats. The inflammatory response was assessed by gross appearance, myeloperoxidase (MPO) activity, tumour necrosis factor α (TNF-α) levels and a histological study of the lesions. We determined prostaglandin E2 production as well as the cyclooxygenases (COX)-1 and -2 expressions by immunohistochemistry and Western blotting. The nuclear factor kappa (NF-κB) p65 and p38 mitogen-activated protein kinase (MAPK) expression levels were also measured by Western blotting. Finally, since PPARγ agonists modulate apoptosis, we tried to clarify its effects under early acute inflammatory conditions. Inflammation following TNBS induction was characterized by increased colonic wall thickness, edema, diffuse inflammatory cells infiltration, necrosis reaching an ulcer index (UI) of 9.66 ± 0.66 cm2 and increased MPO activity and TNF-α colonic levels. Rosiglitazone treatment significantly reduced the morphological alteration associated with TNBS administration and the UI with the highest dose. In addition, the degree of neutrophil infiltration and the cytokine levels were significantly ameliorated. Rosiglitazone significantly reduced the rise in the prostaglandin (PG) E2 generation compared with TNBS group. The COX-1 levels remained stable throughout the treatment in all groups. The COX-2 expression was elevated in TNBS group; however rosiglitazone administration reduced the COX-2 overexpression. A high expression of NF-κB p65 and p38 MAPK proteins appeared in colon mucosa from control TNBS-treated rats; nevertheless, PPARγ agonist treatment drastically decreased them. There were no significant changes in apoptosis after rosiglitazone treatment when compared to TNBS group. In conclusion, rosiglitazone seems to modulate the acute colitis through NF-κB p65 and p38 MAPK signalling pathways.

Introduction

The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors. PPARs regulate gene expression by binding to retinoid X receptors (RXR) as heterodimers. To date, three isoforms, encoded by separated genes, have been identified: PPARα, PPARβ or PPARδ and PPARγ. PPARγ has classically been characterized for its implications in adipocyte differentiation and fat metabolism. Recent studies have shown that thiazolidindiones (TZD) may participate in inflammation control, especially in regulating the production of immunomodulatory and inflammatory mediators (Cabrero et al., 2002, Na and Surh, 2003, Pershadsingh, 2004, Alarcón de la Lastra et al., 2004). Moreover, PPARγ induces a decrease in pro-inflammatory cytokines expression by antagonizing the activities of c-jun-NH2-terminal kinase (JNK) and p38 MAPK in vivo (Desreumaux et al., 2001) and interfering with the transcription factor activation such as nuclear factor NF-κB, signal transducers and transcription activators (STAT), activating protein 1 (AP-1), and the nuclear factor of activated T-cells (NFAT), all of which regulate cytokine gene expression (Dubuquoy et al., 2002).

PPARγ is highly expressed in the colon, mainly in the crypts and surface of epithelial cells (Lytle et al., 2005), as well as in macrophages and T and B lymphocytes (Desreumaux et al., 2001, Lytle et al., 2005); however, its levels are decreased during chronic inflammation in humans and animals (Lefebvre et al., 1999, Katayama et al., 2003).

Ulcerative colitis is a non-specific inflammatory disorder involving primarily the mucosa and submucosa of the colon. Activated immune cells, mainly represented by neutrophils, macrophages, and cytotoxic T cells, play the role of aggressors that attack and destroy the intestinal barrier directly through physical contact or indirectly through the release of reactive oxygen and nitrogen metabolites. Reactive oxygen species are now increasingly recognized to be involved in cell growth, signalling and gene expression (Szanto et al., 2005). Furthermore, reactive oxygen species can activate diverse downstream signalling pathways, such as mitogen-activated protein kinases (MAPKs) or the transcription factor nuclear factor kappa B (NF-κB), thus modulating a number of different steps in the inflammatory cascade. These include production of pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, interferon (INF)-γ IL-12, and IL-6) in different cell-types, degranulation of neutrophils, as well as the expression of important determining parameters of colonic damage i.e. cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) (Dubuquoy et al., 2002, Collino et al., 2006).

There are recent reports which document that PPARγ ligands decrease the degree of inflammation associated with experimental colitis. For example, we have previously reported that the PPARγ ligand, rosiglitazone, was able to suppress the degree of inflammation associated with chronic experimental colitis (Sanchez-Hidalgo et al., 2005). Cuzzocrea et al. (2003) demonstrated the protective effects of the endogenous PPARγ agonist, 15d-PGJ2, on colon damage caused by dinitrobenzene sulfonic acid in the rat. Interestingly, Desreumaux et al. described the effectiveness of PPARγ ligands for the amelioration of 2,4,6,-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis in PPARγ+/− mice. Similarly, Su et al. (1999) and Takagi et al. (2002) reported the attenuation of colonic inflammation by troglitazone in mice. Basing ourselves upon these data there is no doubt that PPARγ may play a central role in anti-inflammatory responses in the colon. Nevertheless, there are many interesting questions regarding the molecular mechanisms for action and signalling pathways in ulcerative colitis. On the other hand, the novelty of this study is that the effects of the TZD, rosiglitazone, in colonic mucosa under acute TNBS-induced colitis in rats were tested for the first time as well as the involvement of the signalling pathways implicated in experimental acute inflammatory disease development. The inflammatory response was assessed by histology and myeloperoxidase (MPO) activity, as an index of quantitative inflammation and neutrophil infiltration in the mucosa. TNF-α production and histological and histochemical analysis lesions were also carried out. Prostaglandin (PG)E2 generation and COX-1, COX-2, nuclear transcription factor NF-κB p65 and MAPK p38 expression were analyzed in order to gain a better insight into the action mechanism(s) of the observed protective effects of rosiglitazone. Finally, since PPARγ agonists have been found to modulate apoptosis in colitis-related colon carcinogenesis (Sanchez-Hidalgo et al., 2005, Kohno et al., 2001) our aim was to study their effects on acute TNBS-induced colitis in rats.

Section snippets

Experimental animals

Male Wistar rats supplied by Animal Services, Faculty of Medicine, University of Seville, Spain, and weighing 180–220 g, were placed in a controlled room (temperature 24–25 °C, humidity 70–75%, lighting regimen of 12L/12D) and were fed a normal laboratory diet (Panlab, Barcelona, Spain). Rats were deprived of food for 24 h prior to the induction of colitis, but were allowed free access to tap water throughout. 14 animals were randomly assigned to each group. Experiments followed a protocol

Protective effects of rosiglitazone in acute TNBS-induced colitis in rats

48 h after intracolonic administration of TNBS, rats showed postration, piloerection and hypomotility. Macroscopic inspection of the cecum, colon and rectum showed evidence of severe colonic mucosal damage, with edema, deep ulcerations and haemorrhage (Fig. 1A). Lesions in the distal colon were quantified using a macroscopic damage score (mean: 9.66 ± 0.66) (Fig. 1B). Control animals underwent severe anorexia with a marked body weight loss compared with the sham animals. A significant increase of

Discussion

In the present study we have demonstrated for first time that the PPARγ ligand, rosiglitazone, attenuated TNBS-induced acute colonic injury in rats. There was an attenuation of morphological signs of cell damage, the colonic mucosa showed ulcers at the beginning of reepithelization and healing process. The decrease in the extent of colitis was accompanied by a lower body weight loss in the animals and a decrease of adhesions between the colon and adjacent organs. The presence of adhesions,

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