Orient Pharm Exp Med (2012) 12:107–111 DOI 10.1007/s13596-012-0055-5
RESEARCH ARTICLE
Mori Folium regulates DSS-induced ulcerative colitis in mice and cytokine production in mast cells In-Young Choi & You-Jeong Kim & Ji-Ye Kee & Min-Chol Kim & Dong-Jin Lee & Sung-One Cho & Jang-Ho Ko & Dae-Seung Kim & Yong-Deok Jeon & Cheol-Hee Yoon & Yun-Jum Park & Jae-Young Um & Seung-Heon Hong
Received: 7 October 2011 / Accepted: 6 January 2012 / Published online: 27 January 2012 # Institute of Oriental Medicine, Kyung Hee University 2012
Abstract Inflammatory bowel disease (IBD) is characterized by detrimental immune reactivity in the gut, and the imbalance between proinflammatory and anti-inflammatory reactivity. The incidence and prevalence rates of IBD in Korea have been increasing rapidly during the past decades. In this study, we investigated regulatory effect of leaf of a mulberry tree (Moraceae) called Gwasang-No.2 in Korea. Leaf of GwasangNo.2 (Gwa.L) inhibited the inflammatory responses on dextran sulfate sodium induced ulcerative colitis in mice and the production of nitrite oxide from lipopolysaccharide and interferon-gamma stimulated mouse peritoneal macrophages. Gwa.L inhibited IL-6 and IL-8 production from HMC-1 cells without cytotoxic effects on cell viability. These results may
In-Young Choi and You-Jeong Kim have equally contributed to this manuscript. I.-Y. Choi : Y.-J. Kim : J.-Y. Kee : M.-C. Kim : D.-J. Lee : S.-O. Cho : J.-H. Ko : D.-S. Kim : Y.-D. Jeon : C.-H. Yoon : S.-H. Hong (*) Department of Oriental Pharmacy, College of Pharmacy and Wonkwang Oriental Medicines Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea e-mail:
[email protected] Y.-J. Park Division of Horticulture and Pet Animal-Plant Science, Wonkwang University, Shinyong-dong, Iksan, Republic of Korea J.-Y. Um College of Oriental Medicine, Kyung Hee University, Hoegi-Dong, Seoul, Republic of Korea
be relevant for future pharmacological or dietary interventions in patients with ulcerative colitis. Keywords Mulberry leaf . Ulcerative colitis . DSS . Nitrite oxide . Anti-inflammation
During the last decades, the incidence of inflammatory bowel disease (IBD) in the Western countries has been increasing. The incidence and prevalence rates of IBD in Korea are still low compared with those of the Western countries, but have been increasing rapidly during the past decades (Yang 2002). Ulcerative colitis (UC) is a chronic, idiopathic IBD that is characterized by bloody diarrhea, colonic mucosal ulceration and, in severe cases, systemic symptoms. An abnormal immune response against antigens of the colonic microbiota in genetically predisposed individuals is suggested to be involved in the etiology of UC (Murakami et al. 2003). Mast cells are innate immune cells that can potentially contribute to IBD through their pro- inflammatory activity and/or effects on immunoregulation. Upon activation, mast cells can immediately release large amounts of pro-inflammatory cytokines that are contained in pre-formed granules (Groschwitz et al. 2009). Mast cell-derived mediators can contribute to colitis severity by enhancing neutrophil influx and thus perpetuating ongoing inflammation (Chichlowski et al. 2010). The genus Morus (commonly known as mulberry) belongs to the family Moraceae, is a group of dioecious woody trees/ shrubs. Many varieties of these species are cultivated on a commercial scale in India, China, Japan and Korea for the sericulture industry (Wakhlu and Singhbhau 2000).
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According to some reports mulberry leaf has functional components such as 1-deoxynojirimycin (DNJ), g-aminobutyric acid (GABA) and flavonoids (Lee et al. 2007). Hence these days mulberry leaf tea is used as functional foods in many countries for prevention of diabetes (Yang and Han 2006) or circulatory problems (Park et al. 2007). Gwasang No.2 is developed as new cultivar of mulberry in Korea. In this study we were interested in the activities of the leaf of Gwasang No. 2 (Gwa.L) on anti-inflammatory response. We used the dextran sulphate sodium (DSS)-induced mouse colitis model and proinflammatory cytokine production from mast cell as the subject for this study. This model resembles human IBD, and is used for pharmacological analysis of potentially effective anti-inflammatory agents (Copper et al. 1993; Camuesco et al. 2005; Ramakers et al. 2007).
Materials and methods
I. Choi et al.
orally for 7 days. The study protocol was approved by the Animal Care and Use Committee of Wonkwang University. Assessment of DSS-induced colitis The mice were checked daily for colitis development by monitoring body weight, gross rectal bleeding, stool consistency and survival. The overall disease severity was assessed by a clinical scoring system on a scale of 0–4 (Copper et al. 1993). In brief, scoring was as follows: 0, no weight loss, no occult blood in the stools and normal stool consistency; 1, weight loss of 1–5%, no occult blood and normal stool consistency; 2, 5–10% weight loss, positive for fecal occult blood and loose stools; 3, 10–20% weight loss, positive for fecal occult blood and loose stools; and 4, greater than 20% weight loss, gross rectal bleeding and diarrhoea.
Preparation of Gwa.L
Peritoneal macrophage cultures
Gwa.L were prepared by decocting the dried leaves with boiling distilled water. The duration of decoction was about 2 h. The decoction was filtered, lyophilized and kept at 4°C. The water extract powder was dissolved in D.W. (100 mg/ml).
TG-elicited macrophages were harvested 2 days after i.p. injection of 2.5 ml TG into mice and then isolated, as reported previously (Xie et al. 1992). Briefly, peritoneal lavage was conducted using 8 ml of HBSS containing 10 U/ml heparin. Next, the cells were distributed into 24well tissue culture plates (3×105 cells/well) in DMEM that was supplemented with 10% heat-inactivated FBS. The cells were then incubated for 3 h at 37°C in an atmosphere of 5% CO2, washed 3 times with HBSS to remove non-adherent cells and then equilibrated with DMEM that contained 10% FBS prior to treatment.
Reagents Thioglycollate (TG) was purchased from Difco Laboratories (Detroit, MI). Iscove’s Modified Dulbecco’s Medium (IMDM), Dulbeccos Modified Eagles Medium (DMEM) containing L-arginine (84 mg/l) and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY). Anti-human IL-6/IL-8 antibody (Ab), biotinylated anti-human IL-6/IL-8 Ab, and recombinant human (rh) IL-6/IL-8 were purchased from R&D Systems (Minneapolis, MN, USA). LPS, PMA, A23187, avidin-peroxidase, 2,2-azino-bis (3ethylbenzthiazoline-6-sulfonic acid), 3- (4,5-dimethylthiazol2-yl)-diphenyl-tetrazolium bromide (MTT), and other reagents were obtained from Sigma (St. Louis, MO, USA). Animals and experimental protocol for colitis Six-week-old female BALB/c mice were purchased from the Da-Mool Science (Taejeon, Korea) and maintained under specific pathogen free conditions at the animal facility of Wonkwang University (Iksan, Korea). To induce experimental colitis, the mice were administered 5% DSS (MW; 36,000– 50,000, MP Biomedicals, Solon OH, USA) dissolved in water that was filter-purified (Millipore Corp., Bedford, MA, USA) for 7 days. The control mice received the filtered water alone. Gwa.L (1 g/kg) or the vehicle (filtered water) was administrated
Measurement of nitrite (NO) concentration Peritoneal macrophages (3×105 cells/well) were pretreated with Gwa.L (0, 0.1, and 1 mg/ml) for 1 h and then cultured with rIFN-γ (10 U/ml) for 6 h, after which they were stimulated with LPS (1 μg/ml) for 48 h. NO synthesis in cell cultures was measured by a microplate assay method as previously described (An et al. 2009). To measure the nitrite concentration, 100 μl aliquots were removed from conditioned medium and incubated with an equal volume of Griess reagent (1% sulfanilamide/0.1% N-(1-naphtyl)-ethylenediamine dihydrochloride/2.5% H3PO4) at room temperature for 10 min. The absorbance at 540 nm was then determined by using a Titertek Multiskan Ascent Reader (Flow Laboratories, North Ryde, Australia). The NO−2 concentration was determined by using sodium nitrite as a standard. This value was determined for each experiment and then subtracted from the value obtained from medium that contained peritoneal macrophages.
Mori Folium regulates DSS-induced ulcerative colitis in mice and cytokine
Culture of HMC-1 cells Human mast cell line, HMC-1, were grown in IMDM medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 10 nM monothioglycerol and 10% heat-inactivated FBS at 37°C in 5% CO2. MTT Assay To test the viability of cells, MTT colorimetric assay was performed as described previously (Choi et al. 2007). HMC1 cells (1×106 cells/ml) were incubated for 8 h after stimulation in the absence or presence of Gwa.L (0, 0.01, 0.1 and 1 mg/ml). After addition of MTT solution, the cells were incubated at 37°C for 4 h. The crystallized MTT was dissolved in dimethyl sulfoxide and measured the absorbance at 540 nm.
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prevented it. Another common feature of the DSS-induced model of colitis is an increase in the DAI (Araki et al. 2006). The DAI of the mice decreased significantly by Gwa.L (1 g/kg) administration (Table 2). The DSS-induced model of colitis is associated with a significant decrease in colon length (Fiocchi 1998; Hendrickson et al. 2002). To assess colon length in the present study, mice from each group were killed at days 7. At days 7 following DSS treatment, we found that colon length in the DSS-administered mice was shorter than that in the Gwa.L-treated mice (Table 3). At the last day of the experiment, body weight of D.W. group, DSS group and Gwa.L group were observed as 18.72 g, 16.58 g and 16.81 g, respectively. Gwa.L seemed to prevent the body weight loss, but there was no significant change.
Effect of Gwa.L on NO production ELISA assay Secreted IL-6 and IL-8 level in supernatants from HMC-1 cells was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s protocol (R&D Systems). Absorption of the avidin-horseradish peroxidase color reaction was measured at 405 nm and compared with serial dilutions of human IL-6 and IL-8 recombinant as a standard.
NO is known a important mediator of inflammatory response. Various studies based on animal models as well as in humans, indicated that NO may be involved in gastrointestinal inflammation and that it may have a pathogenetic role in IBD (Boughton-Smith 1994a, b). Herein we tested the effect of Gwa.L on NO production from activated macrophages. Gwa. L decreased NO production compared with rIFN-γ and LPS treated group (Fig. 1).
Statistical analysis Effect of Gwa.L on cytokine production from HMC-1 cells The results were expressed as mean ± SEM for a number of experiments. Statistical significance was compared between each treated group and control by analysis of variance (ANOVA), with post hoc test of the means according to Tukey’s method. For all tests, P value less than 0.05 was considered significant.
Results Body weight, clinical symptoms and colon length We first observed symptomatic parameters such as body weight loss and disease activity index (DAI) caused by colitis 7 days after starting 5% DSS administration (n06). Table 1 showed administration of 5% DSS caused rectal bleeding however Gwa.L Table 1 Effect of Gwa.L on rectal bleeding mice number in DSSinduced colitis (n06) Rectal Bleeding (n)
Blank (D.W.) DSS+D.W. DSS+Gwa.L(1 g/kg)
We examined the inhibitory effect of Gwa.L on the PMA plus A23187-induced secretion of IL-6 and IL-8 from HMC-1 cells. Culture supernatant was assayed for each cytokine levels by ELISA method. Gwa.L dose-dependently inhibited the secretion of IL-6 (Fig. 2a), and IL-8 (Fig. 2b) in PMA plus A23187-stimulated HMC-1 cells. 1 mg/ml Gwa.L treatment blocked IL-6 and IL-8 production by 42.4±3.25 and 32.6± 6.55% as compared with no treatment of Gwa.L (P
