International Journal of
Molecular Sciences Article
Allyl Isothiocyanate Ameliorates Dextran Sodium Sulfate-Induced Colitis in Mouse by Enhancing Tight Junction and Mucin Expression Min Woo Kim 1 ID , Seungho Choi 1 , Sun Yeou Kim 2 Seung Hyun Oh 2, *,† ID 1
2
* †
ID
, Yeo Sung Yoon 1 , Ju-Hee Kang 2, *,†
ID
and
Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, 08826 Seoul, Korea;
[email protected] (M.W.K.);
[email protected] (S.C.);
[email protected] (Y.S.Y.) Gachon Institute of Pharmaceutical Sciences, Gachon University, 21936 Incheon, Korea;
[email protected] Correspondence:
[email protected] (J.-H.K.);
[email protected] (S.H.O.); Tel.: +82-32-820-4930 (J.-H.K.); +82-32-820-4929 (S.H.O.); Fax: +82-32-820-4829 (J.-H.K. & S.H.O.) These authors contributed equally to this work.
Received: 18 June 2018; Accepted: 9 July 2018; Published: 12 July 2018
Abstract: Inflammatory bowel disease (IBD) is characterized by chronic or recurrent inflammation of the gastrointestinal tract. Even though the current strategies to treat IBD include anti-inflammatory drugs and immune modulators, these treatments have side-effects. New strategies are, therefore, required to overcome the limitations of the therapies. In this study, we investigated the anti-colitic effects of allyl isothiocyanate (AITC), which is an active ingredient present in Wasabia japonica. The DSS-induced colitis model in the mouse was used to mimic human IBD and we observed that AITC treatment ameliorated the severity of colitis. We further studied the mechanism involved to ameliorate the colitis. To investigate the involvement of AITC on the intestinal barrier function, the effect on the intercellular tight junction was evaluated in the Caco-2 cell line while mucin expression was assessed in the LS174T cell line. AITC positively regulated tight junction proteins and mucin 2 (MUC2) against DSS-induced damage or depletion. Our data of in vivo studies were also consistent with the in vitro results. Furthermore, we observed that MUC2 increased by AITC is dependent on ERK signaling. In conclusion, we propose that AITC can be considered as a new strategy for treating IBD by modulating tight junction proteins and mucin. Keywords: allyl isothiocyanate (AITC); Wasabia japonica; intestinal epithelial barrier; tight junction; mucin; mucin 2 (MUC2); goblet cell; dextran sodium sulfate (DSS); colitis
1. Introduction Inflammatory bowel diseases (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC) are characterized by chronic or recurrent inflammation of the gastrointestinal tract [1]. Although the etiology of IBD is poorly understood, a previous study reported that its pathogenesis is related to the dysfunction of the intestinal epithelial barrier [2]. In healthy people, the intestinal epithelial barrier (including the intercellular tight junction and mucus layer) is selectively permeable to the intestinal contents, which prevents various antigens and microorganism from entering the host. In a pathological status of IBD, a dysfunctional intestinal epithelial barrier leads to increased intestinal permeability, which permits the passage of luminal antigens and bacteria and induces a host immune response [3]. A previous human study has reported that IBD patients have a dysfunctional intestinal epithelial barrier with increased intestinal permeability [4]. Int. J. Mol. Sci. 2018, 19, 2025; doi:10.3390/ijms19072025
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The dextran sodium sulfate (DSS)-induced colitis model in the mouse, which is one of the most widely used models for studying IBD, has revealed various aspects of IBD including the pathogenesis, immunology, genetic background, role of microbiome in IBD, and bowel malignancy secondary to IBD [5]. Even though the DSS-induced colitis model is unable to reflect all characters of human IBD, it is widely used because of similar histological features, rapid onset, simplicity, reproducibility, and controllability [6]. In addition, DSS disturbs the intestinal epithelial barrier function by decreasing tight junction proteins and mucin depletion [5,7,8]. The intestinal tight junction protein complexes connects the atypical membranes between intestinal epithelial cells and plays a role as a physical barrier, which provides selective permeability [9]. Defects in the tight junction results in increased gut permeability, which leads to an increased entrance of luminal antigens and contributes to intestinal inflammation [10]. The mucus layer is another physical barrier that protects the intestinal epithelium from harmful agents and bacteria. Mucin, which is the main component of the mucus layer, is produced by the goblet cells and mucin 2 (MUC2) is a major isotype in both human and mouse colon tissue [11,12]. Muc2 knockout mice are more susceptible to DSS-induced colitis. Spontaneous colitis has also been observed without colitis induction [13], which emphasizes the importance of MUC2 for an intestinal barrier function. MUC2 depletion has also been reported as a characteristic of UC patients [14], which indicates that mucin depletion is closely related to human IBD. Irrespective of whether intestinal barrier dysfunction is the primary cause of inflammation or is a consequence secondary to inflammatory cytokines in human IBD, dysfunctional intestinal barrier augments the pathophysiological state of IBD [15]. Therefore, modulating intercellular tight junctions and mucin expression can be effective targets of novel therapeutic strategies for IBD. The cruciferaceae family vegetable Wasabia japonica (WJ) is a popular traditional pungent spice in Asia (including Korea and Japan) and is used to treat rheumatic arthralgia by enhancing the blood circulation and pain relief [16]. It has been reported that a peculiar pungent taste of WJ and other cruciferous plants is derived from the breakdown products of glucosinolates [17]. Glucosinolates have a central thiocyanate group attached to D-glucose and myrosinase converts glucosinolates to glucose and an aglycone, which, in turn, is converted to an isothiocyanate [18]. Various isothiocyanates have been identified in WJ, which is the most abundant allyl isothiocyanate (AITC) [19]. AITC has been reported for its various biological effects including anti-cancer [20], anti-inflammatory [21,22], anti-angiogenic [23], anti-fungal [24], and anti-bacterial effects [25]. In our previous study, we determined that WJ is a functional food that prevents colitis without identifying a specific ingredient that mediates the anti-colitic effect [26]. Davaatseren et al. reported that AITC ameliorates DSS-induced colitis by attenuating angiogenesis and inflammation in mice [27]. Nevertheless, the extent of angiogenesis and inflammation can be affected not only by anti-angiogenic and anti-inflammatory effects of AITC, but also by the severity of the initial intestinal epithelial damage by DSS treatment. To our knowledge, the effects of AITC on the intestinal epithelial have never been explored before. We, therefore, undertook to investigate whether AITC protects the intestinal epithelium from DSS-induced colitis in mice by the modulation of the intestinal barrier including the tight junction and mucin expression. 2. Results 2.1. AITC Ameliorates DSS-Induced Colitis in Mice To investigate whether AITC ameliorates DSS-induced colitis in vivo, AITC (10 mg/kg/day) was administered orally for seven days before DSS treatment. Treated and untreated mice were then exposed to 2.5% DSS in drinking water for seven days (Figure 1A). After sacrifice, the colon tissues were fixed and stained with hematoxylin and eosin (H & E) and microscopically examined. As shown in Figure 1B, 2.5% DSS treatment effectively induced colitis, which is characterized by widely distributed intestinal epithelial damage, goblet cell depletion, inflammatory cell infiltration, and edema. In contrast, the AITC treated group showed a comparatively intact intestinal epithelium
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with a larger number of goblet cells (Figure 1B). To evaluate the severity of DSS-induced colitis, we analyzed analyzed the the colon colon tissues tissues using using aa histological histological scoring scoring system system with with respect respect to to epithelial epithelial cell cell loss, loss, crypt crypt damage, and inflammation [28]. Our results show that the AITC treated group has a significantly damage, and inflammation [28]. Our results show that the AITC treated group has a significantly lower lower score compared with the DSS-only 1C, right). indicate that score compared with the DSS-only treatedtreated group group (Figure(Figure 1C, right). These These resultsresults indicate that AITC AITC treatment ameliorates the severity of DSS-induced colitis. Furthermore, we aobserved a treatment ameliorates the severity of DSS-induced colitis. Furthermore, we observed significant significant difference in the surface epithelial cell loss between the AITC treated group and the DSSdifference in the surface epithelial cell loss between the AITC treated group and the DSS-only treated only (Figure 1C,indicates left), which AITCthe may protectepithelium the intestinal epithelium grouptreated (Figuregroup 1C, left), which that indicates AITC maythat protect intestinal through direct through direct effects on the epithelium regardless of its anti-inflammatory effects and/or antieffects on the epithelium regardless of its anti-inflammatory effects and/or anti-angiogenic effects. angiogenic effects. We, therefore, hypothesized that AITC has a protective effect on DSS-induced We, therefore, hypothesized that AITC has a protective effect on DSS-induced colitis by regulating the colitis by regulating the intestinal epithelial barrier. intestinal epithelial barrier.
Figure 1. Protective effects of AITC against DSS-induced colitis in the mouse. (A) Animal experiment design for AITC treatment and colitis induction; induction; (B) Histological Histological figure figure of colon stained stained with with H & E (Scale bar 100 μm); and (C) To evaluate severity of DSS-induced colitis, a histological scoring system µm); evaluate mean ± ± standard was used. Numerical data were shown as aa mean standard deviation deviation (t-test (t-test was used for statistical 0.01). analysis, ** p
