Oral nitrite ameliorates dextran sulfate sodium-induced acute experimental colitis in mice

Abstract

Inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis are chronic inflammatory disorders of the intestinal tract with excessive production of cytokines, adhesion molecules, and reactive oxygen species. Although nitric oxide (NO) is reported to be involved in the onset and progression of IBDs, it remains controversial as to whether NO is toxic or protective in experimental colitis. We investigated the effects of oral nitrite as a NO donor on dextran sulfate sodium (DSS)-induced acute colitis in mice. Mice were fed DSS in their drinking water with or without nitrite for up to 7 days. The severity of colitis was assessed by disease activity index (DAI) observed over the experimental period, as well as by the other parameters, including colon lengths, hematocrit levels, and histological scores at day 7. DSS treatment induced severe colitis by day 7 with exacerbation in DAI and histological scores. We first observed a significant decrease in colonic nitrite levels and increase in colonic TNF-α expression at day 3 after DSS treatment, followed by increased colonic myeloperoxidase (MPO) activity and increased colonic expressions of both inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) at day 7. Oral nitrite supplementation to colitis mice reversed colonic nitrite levels and TNF-α expression to that of normal control mice at day 3, resulting in the reduction of MPO activity as well as iNOS and HO-1 expressions in colonic tissues with clinical and histological improvements at day 7. These results suggest that oral nitrite inhibits inflammatory process of DSS-induced experimental colitis by supplying nitrite-derived NO instead of impaired colonic NOS activity.

Introduction

Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders of the intestinal tract with excessive production of cytokines, adhesion molecules, and reactive oxygen species (ROS) [1]. The etiology of IBDs is believed to involve inappropriate host responses to the complex commensal microbial flora in the gut resulting from mucosal barrier dysfunction, such as an abnormal leaky mucus layer, altered tight junction protein expression for distribution, and increased epithelial apoptosis [2].

In order to study etiology of IBDs, mouse experimental colitis induced by oral administration of dextran sulfate sodium (DSS) is widely used as a standardized colitis model [3]. Histologically, colitis induced by DSS is characterized by infiltration of inflammatory cells into the lamina propria, accompanied by lymphoid hyperplasia, focal crypt damage, and epithelial ulceration [4]. DSS has a direct toxic effect on the epithelium and destroys the mucosal barrier, allowing bacteria to come into contact with lamina propria cells [5], resulting in activation of intestinal macrophages and secreting pro-inflammatory cytokines such as TNF-α [6]. These innate immune dysfunctions mediated by intestinal macrophage induce excessive production of superoxide, leading to further inflammatory process and endothelial dysfunction as a result of ability of superoxide to inactivate endogenous NO [7].

There is growing experimental evidence that nitric oxide (NO), enzymatically generated by three isoforms of nitric oxide synthase (NOS) (endothelial NOS: eNOS, neuronal NOS: nNOS, and inducible NOS: iNOS), plays an important role in the intestinal barrier and immune functions. Impaired NO production in intestine, therefore, might have a causative effect on the progression and sequelae of IBDs. Although controversy has continued as to whether the in vivo effects of iNOS-induced NO are beneficial or detrimental on the experimental colitis [8], the majority of studies using selective inhibitors of iNOS and iNOS-deficient mice have shown improvement in experimental colitis, suggesting a possible involvement of inflammatory iNOS in the progression of IBDs. On the other hand, constitutively produced NO by eNOS and nNOS appears to be a homeostatic regulator of numerous essential functions of the gastrointestinal mucosa, such as maintenance of adequate perfusion, and regulation of microvascular and epithelial permeability. The protective actions of constitutive NOS also include reduction of leukocyte adherence, inhibition of macrophage activation, and inhibition of Th1 type cytokines by inactivation of cytokine processing. These evidences suggest protective effects of NO produced by constitutive NOS on the pathogenic processes of acute colitis following barrier dysfunction.

Thus, while the association of NOS-mediated NO dynamics with pathophysiology of IBDs has been investigated in detail, exogenous NO donors are attempted to be applied to experimental colitis models as a therapeutic intervention [9], [10]. Salas et al. showed that subcutaneous administration of NO donor modulates DSS-treated colitis in mice, suggesting that the beneficial effect of NO is related to a reduction in leukocyte recruitment and subsequent pro-inflammatory cytokine production by inhibiting endothelial adhesion molecule expression in the early phase of this animal model [9].

Recently, an alternative pathway fundamentally different from enzymatic NOS system for NO generation was discovered in which nitrite and nitrate are reduced in vivo to form NO, exerting physiological functions in the gastrointestinal tract and cardiovascular system [11], [12]. Moreover, nitrate and nitrite are thought to have therapeutic application for the diseased states where oxygen availability is reduced or NOS activity is decreased [13], [14], [15].

Based on these recent observations, we hypothesized that oral nitrite supplementation could be effective on reducing inflammatory processes in DSS-treated colitis mice, which mimics IBDs in that it induces inflammation and macrophage activation accompanied with disrupted epithelial barrier integrity [16]. Since DSS-treated colitis by 5–10 days in mice is considered to be an acute colitis model [17], we investigated how constitutive NOS and iNOS are implicated in the onset and progression of DSS-treated mice colitis, and also investigated the effect of oral nitrite on clinical and histological improvement following inflammatory responses including colonic TNF-α, iNOS and HO-1 expressions especially focusing on the role of NO availability in the early phase of this model.