Mori Folium regulates DSS-induced ulcerative colitis in mice and cytokine production in mast cells
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- Choi, I., Kim, Y., Kee, J. et al. Orient Pharm Exp Med (2012) 12: 107. doi:10.1007/s13596-012-0055-5
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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 Gwasang-No.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 be relevant for future pharmacological or dietary interventions in patients with ulcerative colitis.
KeywordsMulberry leafUlcerative colitisDSSNitrite oxideAnti-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).
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
Preparation of Gwa.L
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).
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 (3-ethylbenzthiazoline-6-sulfonic acid), 3- (4,5-dimethylthiazol-2-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 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.
Peritoneal macrophage cultures
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 24-well 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.
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.
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.
To test the viability of cells, MTT colorimetric assay was performed as described previously (Choi et al. 2007). HMC-1 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.
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.
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.
Body weight, clinical symptoms and colon length
Effect of Gwa.L on rectal bleeding mice number in DSS-induced colitis (n = 6)
Rectal Bleeding (n)
Effect of Gwa.L on DAI in DSS-induced colitis (n = 6)
0 ± 0
3.0 ± 0.37
1.28 ± 0.35
Effect of Gwa.L on colon length in DSS-induced colitis (n = 6)
6.9 ± 0.39
5.5 ± 0.36
5.8 ± 0.11
Effect of Gwa.L on NO production
Effect of Gwa.L on cytokine production from HMC-1 cells
In this study, we showed that administration of Gwa.L prevented body weight loss and an increase in DAI scores in mice with DSS-induced colitis. Furthermore, Gwa.L alleviated the symptoms of DSS-induced colitis and improved colitis-induced morphological damage by preventing the colon length. Gwa.L also inhibited the production of NO from activated macrophages and IL-6/IL-8 production from mast cells.
According to some reports mulberry leaf has functional components such as 1-deoxynojirimycin (DNJ), γ-aminobutyric acid (GABA) and flavonoids. So, mulberry leaf protect rat tissues from immobilization stress-induced inflammation (Lee et al. 2007) and oxidant damages, atherogenesis effect (Yen et al. 1996).
Hence these days mulberry leaf tea is used as functional foods in many countries for prevention of diabetes or circulatory problems. Gwasang No.2 is developed as new cultivars of mulberry in Korea. We tested the activity of Gwa.L to suggest a new candidate for functional food or remedy.
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. NO concentrations in colons were found to be more than 100 times higher in the UC patients than in the controls (Lundberg et al. 1994). In this study we showed Gwa.L inhibited the NO production from activated macrophages. The mast cell count is significantly higher in the colonic mucosa of UC patients than in that of patients with Crohn’s disease, Th1 associated inflammatory bowel disease (Nolte et al. 1990; Sasaki et al. 2002). Mast cell-derived mediators can contribute to colitis severity by enhancing neutrophil influx and thus perpetuating ongoing inflammation (Chichlowski et al. 2010). Cytokine and chemokine mRNA expression profiles in UC have been characterized in former studies (Autschbach et al. 2002; Sartor 1994; Gotteland et al. 1999). IL-6 play roles in mediating the progression of many inflammatory diseases. Recently, humanized anti-human IL-6 receptor monoclonal antibody has been clinically developed as a therapeutic agent for some autoimmune inflammatory diseases, such as rheumatoid arthritis and Crohn’s disease, originally in Japan (Nishimoto 2005). IL-8 concentrations in dialysates from patients with active UC were significantly higher than in controls and correlated with disease activity. anti-IL-8 antibody partially (50%) inhibited these stimulatory effects of UC dialysates (Keshavarzian et al. 1999). There was a statistically significant positive correlation between CXCL8 (IL-8) with clinical activity index and endoscopic activity index in UC (Zahn et al. 2009). We found that Gwa.L inhibited IL-6 and IL-8 production from activated mast cells by 42.4 ± 3.25 and 32.6 ± 6.55%.
In conclusion, Gwa.L improved histological scores, maintains the colon architecture, and decreases the release of inflammatory mediators in DSS-induced colitis in mice. Our findings may be relevant for future pharmacological or dietary interventions in patients with ulcerative colitis. These results can apply to produce the health and functional foods that have modulating effects for these diseases.