Oral nitrite ameliorates dextran sulfate sodium-induced acute experimental colitis in mice
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.
Section snippets
Animals
Specific pathogen-free male ICR (CD-1) mice, 7 week old, weighing 30–35 g, from Kiwa Laboratory Animals (Wakayama, Japan) were allowed food (CE-2, CLEA Japan) and reverse osmosis (RO) water ad libitum, and were kept on a 12/12 h light/dark cycle with at least 7 days of local vivarium acclimatization before experimental use. All the protocols were approved by the Institutional Animal Care and Use Committee at the University of Josai Life Science Center and were consistent with the Guide for the Care
Characteristics of DSS-treated colitis
As described in the experimental procedures in the methods, DSS-treated mice drink 15% more water than DSS + NaNO2-treated mice. Therefore, DSS + NaNO2-treated mice were given 3.0% DSS to provide the same total dose of DSS between them. We calculated the average intake of DSS in DSS-treated and DSS + NaNO2-treated mice (245.6 ± 28.4 mg/mouse/day and 248.6 ± 11.1 mg/mouse/day, respectively) resulting in no significant difference in DSS intake between the two groups (Table 1). No difference of average intake
Discussion
Constitutive NO production by eNOS prevents the accumulation of platelets and adherent leukocytes, thus creating a non-thrombogenic environment in the vasculature. In the intestinal tract, small amounts of NO produced by constitutive forms of NOS (eNOS and nNOS) are thought to be physiologic and protective by regulating mucosal integrity, especially in response to noxious stimuli. Because acute DSS-induced colitis represents primarily a barrier disruption model which results in the activation
Acknowledgments
The authors would like to thank Yoshinori Kubo, Masafumi Saito, Yukari Takahashi, and Sonoko Harada for their technical assistance.
References (51)
- et al.
A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice
Gastroenterology
(1990) - et al.
Nitric oxide supplementation ameliorates dextran sulfate sodium-induced colitis in mice
Lab. Invest.
(2002) - et al.
Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response
Free Radic. Biol. Med.
(2009) - et al.
Dietary nitrite inhibits early glomerular injury in streptozotocin-induced diabetic nephropathy in rats
Nitric Oxide
(2007) - et al.
Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker
J. Invest. Dermatol.
(1982) - et al.
Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis
J. Biol. Chem.
(2007) - et al.
Increased disease activity in eNOS-deficient mice in experimental colitis
Free Radic. Biol. Med.
(2003) - et al.
Nitric oxide synthase distribution in the rat intestine: a histochemical analysis
Gastroenterology
(1993) - et al.
Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation
J. Biol. Chem.
(2000) - et al.
The nitric oxide congener nitrite inhibits myeloperoxidase/H2O2/Cl−-mediated modification of low density lipoprotein
J. Biol. Chem.
(2001)
Role of blood- and tissue-associated inducible nitric-oxide synthase in colonic inflammation
Am. J. Pathol.
Current view of the immunopathogenesis in inflammatory bowel disease and its implications for therapy
World J. Gastroenterol.
Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis
FASEB J.
Increased permeability in dextran sulphate colitis in rats: time course of development and effect of butyrate
Scand. J. Gastroenterol.
Histological analysis of murine colitis induced by dextran sulfate sodium of different molecular weights
Exp. Anim.
The ICE inhibitor pralnacasan prevents DSS-induced colitis in C57BL/6 mice and suppresses IP-10 mRNA but not TNF-α mRNA expression
Dig. Dis. Sci.
Colonic blood flow responses in experimental colitis: time course and underlying mechanisms
Am. J. Physiol.
Nitric oxide in inflammatory bowel disease
Inflamm. Bowel Dis.
NCX-1015, a nitric-oxide derivative of prednisolone, enhances regulatory T cells in the lamina propria and protects against 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice
Proc. Natl. Acad. Sci. USA
The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics
Nat. Rev. Drug Discov.
NO generation from inorganic nitrate and nitrite: role in physiology, nutrition and therapeutics
Arch. Pharm. Res.
Dietary nitrite supplementation protects against myocardial ischemia–reperfusion injury
Proc. Natl. Acad. Sci. USA
Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction
Am. J. Physiol.
Intestinal barrier dysfunction in inflammatory bowel diseases
Inflamm. Bowel Dis.
Animal models of inflammatory bowel disease
J. Gastoroenterol.
Cited by (35)
Nitrate ameliorates dextran sodium sulfate-induced colitis by regulating the homeostasis of the intestinal microbiota
2020, Free Radical Biology and MedicineNitrate from diet might fuel gut microbiota metabolism: Minding the gap between redox signaling and inter-kingdom communication
2020, Free Radical Biology and MedicineDietary nitrite supplementation attenuates cardiac remodeling in L-NAME-induced hypertensive rats
2017, Nitric Oxide - Biology and ChemistryCitation Excerpt :Protein samples were obtained by centrifugation at 10,000 g for 5 min following methanol precipitation (tissue sample:methanol = 1:2 wt/volume, plasma:methanol = 1:1 vol/vol, 4 °C). Nitrite and nitrate concentrations in the plasma and heart were measured using a dedicated HPLC system (ENO-20; Eicom, Kyoto, Japan) [11]. The detection method was based on the separation of nitrite and nitrate by ion chromatography, followed by the on-line reduction of nitrate to nitrite, post-column derivatization with a Griess reagent, and detection at 540 nm.
LED measurement for development of a non-destructive detector of unsuitable chicken eggs in influenza vaccine production
2015, Biosystems EngineeringCitation Excerpt :585 nm is the absorbance range of haemoglobin (Dover, Sadick, & Goldman, 1999). 635 nm is the absorbance range of methaemoglobin (Ohtake et al., 2010). Unsuitable eggs were identified using spectral data obtained from 50 normal eggs and 50 unsuitable eggs.
S-nitrosylation of mouse galectin-2 prevents oxidative inactivation by hydrogen peroxide
2015, Biochemical and Biophysical Research CommunicationsDietary nitrite supplementation improves insulin resistance in type 2 diabetic KKA<sup>y</sup> mice
2015, Nitric Oxide - Biology and Chemistry