Hydrogen mediates suppression of colon inflammation induced by dextran sodium sulfate

https://doi.org/10.1016/j.bbrc.2009.05.117 Get rights and content

Abstract

By its antioxidant effect, molecular hydrogen gas (H2) was reported to protect organs from tissue damage induced by ischemia reperfusion. To evaluate its anti-inflammatory effects, we established a mouse model of human inflammatory bowel disease (IBD) by supplying mice with water containing (1) dextran sodium sulfate (DSS) (5%), (2) DSS (5%) and H2, or (3) H2 only ad libitum up to 7 days. At day-7, DSS-induced pathogenic outcomes including, loss of body weight, increase of colitis score, pathogenic shortening of colon length, elevated level of IL-12, TNF-α and IL-1β in colon lesion, were significantly suppressed by the addition of H2 to DSS solution. Histological analysis also revealed that the DSS-mediated colonic tissue destruction accompanied by macrophage infiltration was remarkably suppressed by H2. Therefore, the present study indicated that H2 can prevent the development of DSS-induced colitis in mice.

Introduction

Molecular hydrogen (H2) is recognized as possessing antioxidative effects. Previous studies demonstrated that H2 in the form of gas or dissolved in water can suppress tissue injuries caused in brain, liver, and heart by oxidative stress from ischemia reperfusion [1], [2], [3]. Unlike other gaseous molecules, such as NO or O2, the small H2 molecule can penetrate solid substances, even plastic. Supported by such rapid permeability, while precise chemical mechanism by which H2 quenches oxidative stress is to be elucidated [4], ingestion of H2-water which holds neutral pH is thought to function as a strong antioxidant agent because of its extremely negative redox potential value [5]. However, the effects of H2 on inflammation induced by mechanisms other than ischemia reperfusion remain to be elucidated.

Dextran sodium sulfate (DSS)-induced rodent colitis has been reported as an animal model of human inflammatory bowel disease (IBD), especially ulcerative colitis [6], [7]. DSS is a sulfated polysaccharide that interferes with epithelial cell barrier function. This exposes the lamina propria to luminal bacterial antigens, which, in turn, elicits the activation of innate immunity [8], [9]. When applied to mice in drinking water, DSS induces colitis, which is characterized by weight loss, diarrhea and/or grossly bloody stool, and the histopathological features of intestinal inflammation, i.e., erosion of crypt [10].

Impaired antioxidant mechanism is implicated as one of pathogenic causes of DSS-induced colitis [11]. Since both inflammation and oxidation processes are reciprocally related [12], [13], [14], as such, antioxidant effects of H2 should prevent its development by suppressing proinflammatory cytokines, e.g., IL-1β, IL-12, and TNF-α, expressed in the colitis lesion [15], [16], [17]. Especially, these proinflammatory cytokines are considered to be responsible for the tissue destructions occurring in the DSS-induced mouse colitis as well as human IBD [6], [18], [19]. However, since it is unclear if ingestion of H2-water can efficiently prevent or suppress the inflammatory outcome of DSS-induced colitis, we examined the effects of molecular H2 dissolved in water on DSS-induced mouse colitis.

Animals. BALB/c mice (8–10 w old males, n = 6/group) were caged in specific pathogen-free (SPF) conditions. The animals were kept in a conventional room with a 12-h light-dark cycle at constant temperature. The experimental procedures employed in this study were approved by the Forsyth IACUC.

Measurement of molecular hydrogen. Molecular hydrogen (H2) present in water or organs of mice was measured using a needle-type Hydrogen Sensor (Unisense A/S, Aarhus, Denmark) following the method published by Hayashida et al. [2].

Generation of H2 dissolved water. High purity H2 gas (Airgas East, Salem, NH) was injected into water, Ringer’s solution, or 5% DSS in water until H2 concentration reached saturation (0.78 mM, at 25 °C).

Induction of DSS-induced colitis. On Day-0, control regular distilled water, DSS (5% [wt/vol], 30–40 kDa; Acros Organics, Morris Plains, NJ) dissolved in distilled water with or without saturated H2 or distilled water with saturated H2 only were applied to mice ad libitum as drinking water in a feeding glass bottle (Schott Duran, Mainz, Germany) with rubber top and metal tube. The biophysical property of fresh water containing saturated H2 showed (1) H2 concentration, 0.78 mN, (2) pH 7.43  7.76, and (3) ORP −462 to −511 mV. After 24 h, these values were measured as (1) H2 concentration, 0.39  0.42 mM, (2) pH 7.34  7.63, and (3) ORP −388∼−420 mV. Fresh DSS solution, with or without hydrogen, and hydrogen-only water was prepared daily. Mice were monitored on a daily basis for the next 7 days to measure body weight and colitis score. After 7 days, mice were sacrificed, and colonic tissues were collected. The colon was removed at the two positions closest to the ileocecal valve and the rectum, and the length was measured. Sections (1 cm) of the distal and proximal colon were fixed and embedded in OCT compound for histological analysis. The remaining part of the colon was weighed and frozen in liquid nitrogen for detection of cytokines.

Colitis score. The colitis score was assessed daily during DSS induction by trained individuals blinded to the treatment groups [20]. The baseline colitis score was determined on day 0. Briefly, no weight loss was scored as 0; weight loss of 1–5% from baseline as 1; 5% to 10% as 2; 10% to 20% as 3; and more than 20% as 4. For stool consistency, a score of 0 was assigned for well-formed pellets, 2 points for pastey and semi-formed stools that did not adhere to the anus, and 4 points for liquid stools that did adhere to the anus. For bleeding, a score of 0 was assigned for no blood, 2 points for positive hemoccult, and 4 points for gross bleeding. These scores were added together and divided by three, resulting in a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis).

Histological evaluation. Tissue sections (8 μm) of distal portion of transversal colon were stained with hematoxylin and eosin (H & E). Histological scoring was performed based on amount and depth of inflammation and amount of crypt regeneration or damage [21] Briefly, scores were graded as follows: (1) Inflammation amount: none, 0; slight, 1; moderate, 2; severe, 3 (2) Inflammation depth: none, 0; mucosa, 1; mucosa and submucosa, 2; transmural, 3 (3) Crypt damage: none, 0; basal 1/3 damaged, 1; basal 2/3 damaged, 2; only surface epithelium intact, 3; entire crypt and epithelium lost, 4. Sections were scored for each feature separately, and the scores were added to reach the final histological scoring for individual colon specimens.

Immunofluorescent identification of F4/80-positive macrophages. Sectioned colon tissues were fixed with a mixture of acetone (50%) and methanol (50%). Macrophages present in the colon were stained with anti-F4/80 MAb conjugated with biotin (Rat IgG2b, AbD Serotec, Oxford, UK) followed by FITC-Avidin (BD Pharmingen, San Diego, CA). Irrelevant Rat MAb conjugated with biotin (BD Pharmingen) was used as control. The staining pattern was analyzed at ×400 magnification using a Leica TCS/SP-2 laser scan confocal microscope.

ELISA. Dissected transversal colonic tissues were homogenized with a Dounce glass homogenizer in PBS supplemented with 0.05% Tween 20, phenylmethyl sulfonyl fluoride (1 mM; Sigma, St. Louis, MO) and protease inhibitor cocktail (Sigma), followed by centrifugation for 10 min at 18,000 rpm. The resulting supernatant was subjected to ELISA for the measurement of TNF-α (Mouse TNF-α ELISA MAX™ Set, Biolegend, San Diego, CA), and IL-1β or IL-12p40 (Murine ELISA Development kit, Peprotech, Rocky Hill, NJ, respectively).

Section snippets

Results

Inhalation of H2 gas can suppress oxidation-dependent tissue injury of the small intestine caused by ischemia reperfusion which was mediated by syngeneic small intestinal transplantation [22]. While this indicates that administration by gas can reach the small intestine, it is unclear whether H2-dissolved water applied orally can affect the concentration of H2 in the colon. Efficacy would depend on the interplay between H2 produced or absorbed by intestinal bacteria and exogenously supplied H2.

Discussion

The present study demonstrated that H2 can attenuate DSS-induced colitis by down-regulating the expression of proinflammatory cytokines, as well as suppressing the infiltration of macrophages in the colon lesion. The administration of H2 remarkably reduced the clinical symptoms of DSS-induced colitis, i.e., body weight loss, visible fecal blood and diarrhea, colitis score, and shortening of colon length. Histopathological evaluation further supported the effects of H2 on the prevention of

Acknowledgments

We thank Dr. James G. Fox (Division of Comparative Medicine and Biological Engineering, Massachusetts Institute of Technology, Boston, MA) for his cooperation in designing of animal protocol used this study. This study was supported by a research grant from Skyview Enterprises.

References (28)

  • K.C. Wood et al.

    The hydrogen highway to reperfusion therapy

    Nat. Med.

    (2007)
  • G. Rogler et al.

    Cytokines in inflammatory bowel disease

    World J. Surg.

    (1998)
  • M.L. Clapper et al.

    Dextran sulfate sodium-induced colitis-associated neoplasia: a promising model for the development of chemopreventive interventions

    Acta Pharmacol. Sin.

    (2007)
  • H. Tlaskalova-Hogenova et al.

    Involvement of innate immunity in the development of inflammatory and autoimmune diseases

    Ann. NY Acad. Sci.

    (2005)
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