nutrients Article
Ginseng Berry Extract Attenuates Dextran Sodium Sulfate-Induced Acute and Chronic Colitis Wei Zhang 1 , Li Xu 1 , Si-Young Cho 2 , Kyung-Jin Min 3 , Tatsuya Oda 4 , LiJun Zhang 1 , Qing Yu 5,6 and Jun-O Jin 1, * 1 2 3 4 5 6
*
Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, Shanghai 201508, China;
[email protected] (W.Z.);
[email protected] (L.X.);
[email protected] (L.Z.) R & D Unit, AmorePacific Corporation, 1920 Yonggudae-ro, Giheung-gu, Yongin-si, Gyeonggi-do 17074, Korea;
[email protected] Department of Biological Sciences, Inha University, Incheon 22212, Korea;
[email protected] Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 55001, Japan;
[email protected] Department of Immunology and Infectious Diseases, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA;
[email protected] Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA 02115, USA Correspondence:
[email protected]; Tel.: +86-131-2282-1851
Received: 6 January 2016; Accepted: 29 March 2016; Published: 5 April 2016
Abstract: This study investigates the in vivo functions of ginseng berry extract (GB) as a therapy for dextran sodium sulfate (DSS)-induced colitis. C57BL/6 mice were given drinking water containing DSS (3%) for eight days to induce acute colitis. At the same time, the mice received an oral dose of GB (50 mg/kg) once daily. The GB-treated mice were less susceptible to the development of acute colitis than were control mice treated with saline, as determined by weight loss, disease activity, and colon histology. The administration of GB to DSS-treated mice also reduced the numbers and inhibited the activation of colon-infiltrating T cells, neutrophils, intestinal CD103´ CD11c+ dendritic cells (cDCs), and macrophages. In addition, GB treatment promoted the migration of CD103+ CD11c+ cDCs and expansion of Foxp3+ regulatory T cells in the colons of DSS-treated mice. Similarly, in the DSS-induced chronic colitis model, GB treatment improved the macroscopic and histological appearance of the colon wall when compared to untreated control mice, as indicated by longer colon length and lower histological scores. This is the first report to show that oral administration of GB suppresses immune activation and protects against experimentally induced colitis. Keywords: ginseng berry extract; mouse colitis; intestinal dendritic cell; intestinal macrophage
1. Introduction Ginseng root has been used in Asian countries as a traditional medicine for various diseases, such as cancer, autoimmune disorders, and atherosclerosis [1]. Previous reports have shown that ginseng root extract (GR) has strong anti-inflammatory properties [2,3] and can attenuate dextran sodium sulfate (DSS)-induced colitis and colon cancer in mice [4]. Ginseng berry extract (GB) shows similar anti-inflammatory effects and can ameliorate streptozotocin-induced diabetes in mice [5]. The ginsenosides in GR are the active components and contribute greatly to its pharmacological activities [6]. Previous studies have shown that the ginsenoside profile of GB differs from that of GR, and that the ginsenoside content is four to six times higher in GB than in GR [6,7]. Moreover, GB shows a greater potency than GR in inducing anti-hyperglycemic activities [8] and relaxation effects on the penile corpus cavernosum smooth muscle [7]. However, the in vivo anti-inflammatory effects of GB, and especially on DSS-induced colitis, have not been investigated or compared to the effects of GR. Nutrients 2016, 8, 199; doi:10.3390/nu8040199
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The etiology of inflammatory bowel diseases (IBDs), such as ulcerative colitis (UC) and Crohn’s disease (CD), still remains unknown. Interactions between immunologic, genetic, and environmental factors and the intestinal microflora may contribute to the pathogenesis of IBDs [9]. Dysregulated immune responses in the intestinal mucosa cause an overproduction of pro-inflammatory cytokines, such as IL-6, IL-17, IFN-γ, and TNF-α, and contribute to the development of colitis [10,11]. Immune cells, including dendritic cells (DCs), macrophages, neutrophils, and T cells, together with the pro-inflammatory cytokines, collaborate to perpetuate and sustain the inflammatory responses in the colon, eventually leading to tissue damage and the development of colitis [12]. Inhibition of myeloid cell migration in IL-10-knockout mice and depletion of phagocytes ameliorate the development of colitis, suggesting that DCs and macrophages are directly involved in the development of this disease [13]. The DCs isolated from the colon tissue of patients with UC show higher CD40 expression than is observed in DCs from healthy individuals [14]. Moreover, depletion of DCs before the induction of colitis by DSS results in exacerbation of the disease [15,16]. In addition, intestinal CD103+ DC subsets are able to induce expansion of Foxp3-expressing regulatory T (Treg) cells [9,15], which prevent inflammation in the colon [17]. By contrast, CD103´ DCs promote the generation of T helper (Th) 17 cells [15,18], which enhances the pathogenesis of UC [19]. Thus, the protective/pathogenic role of distinct DC subsets in the intestine remains an active area of investigation. In this study, we investigated the effect of GB on DSS-induced acute and chronic colitis models and tested the hypothesis that GB has anti-inflammatory and immune-suppressing effects that protect against these diseases. 2. Materials and Methods 2.1. Animals C57BL/6 mice were purchased from Shanghai Public Health Clinical Center and kept under pathogen-free conditions. The mice were maintained in a room with controlled temperature (20–22 ˝ C), humidity (50%–60%), and light (12 h:12 h) and were given free access to standard rodent chow and water. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Shanghai Public Health Clinical Center. The protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University (Permit Number: SYXK-2010-0098). Mice were sacrificed by CO2 inhalation euthanasia, and all efforts were made to minimize suffering. 2.2. Chemicals DSS (molecular weight 36,000–50,000) was purchased from MP Biomedicals (Guang Zhou, China). Freshly harvested four-year-old Korean ginseng (Panax ginseng CA Meyer) roots and berries, cultivated in the Chungbuk Province of Korea, were purchased in July 2014, and the species identity was confirmed by Dr. Ki Ho Kim from Biolandkorea Co. (Chungnam, Korea). Voucher specimens (GBP1207 for ginseng berry) were deposited at the herbarium of the College of Environmental and Bioresource Sciences, Chonbuk National University, Korea. Ginseng roots (100 g) were extracted with 600 mL 55% ethanol at 65–68 ˝ C for 24 h, with stirring. This extraction procedure was repeated six times. The pooled filtrates were vacuum evaporated at 50–60 ˝ C to yield 50 g of solid content, which was further purified with 500 mL 88% ethanol at 5–10 ˝ C overnight and centrifuged at 5000ˆ g for 10 min. The supernatants were evaporated and spray dried to yield 15 g of ginseng extract [20]. The seeds of the ginseng berries were separated and removed, and the remnants were dried in a hot air stream and then refluxed with 70% ethanol for 10 h. The extract was filtered, concentrated under reduced pressure at 45 ˝ C, and lyophilized to produce a GB powder, which was stored at ´20 ˝ C until use. The endotoxin levels in GR and GB were evaluated using a Limulus amebocyte lysate (LAL) assay kit (Lonza). The GR and GB used in all experiments contained less than 0.1 endotoxin unit/mL.
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2.3. Comparison and Standardization of Ginsenoside Composition Minerals were determined by digesting the ash from ginseng roots and berries with 3M hydrochloric acid, followed by atomic absorption spectrophotometry analysis for calcium (Ca), magnesium (Mg), zinc (Zn), and iron (Fe) and the flame photometry analysis for potassium (K) [21]. Soluble and insoluble vitamins were determined by HPLC. Vitamins (Vits.) B1, B2, B6, folic acid, pantothenic acid, and niacin were separated on a SUPELCO Discovery C18 column (150 ˆ 4.6 mm, ID 5 µm) at 35 ˝ C and detected by PDA at 220 nm using a mobile phase of K2 HPO4 /methyl alcohol (99/1) at a 1 mL/min flow rate and column pressure of 92 kgf [22]. The insoluble Vits, such as Vit. A, E and K, were separated using n-hexane/isopropanol (99:1) as a mobile phase at a flow rate of 1 mL/min on a Lichrosorb Si60 (250 ˆ 4 mm, ID 5 µm) column at 35 ˝ C [23]. 2.4. DSS-Induced Acute Colitis C57BL/6 mice were given 3% DSS in their drinking water for eight consecutive days. The drinking water was changed daily, according to the water volume the mice had consumed during the previous day. Mice were orally administered 50 mg/kg/100 µL of GB, GR, or 0.9% saline (control) during the DSS treatment. The mice in different treatment groups were gender- and age-matched. The mice were sacrificed on day 8 and colonic tissues were harvested for analysis. The disease activity index (DAI) was calculated, as previously described [24]. In brief, the DAI was scored on a scale from 0 to 4 using the following parameters: loss of body weight (0, normal; 1, 0%–5%; 2, 5%–10%; 3, 10%–20%; 4, >20%), stool consistency (0, normal; 2, loose stools; 4, watery diarrhea), and the occurrence of gross blood in the stool (0, negative; 4, positive). 2.5. DSS-Induced Chronic Colitis Chronic colitis was induced by three-cycle administrations of DSS-containing drinking water, with a modification to a previously described method [25]. C57BL/6 mice received 2.5% DSS drinking water for six days, followed by two days of regular drinking water. These mice continued to receive 2.5% DSS drinking water during days 8–14 and 16–22. Mice with chronic DSS colitis were orally treated with GB (50 mg/kg) or 0.9% saline daily on days 1–6, 8–14, and 16–22. The mice in different treatment groups were gender- and age-matched. The mice were sacrificed on day 22 and colon tissue was harvested for further experiments. 2.6. Hematoxylin and Eosin Staining As described in detail previously [26], colon samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm thickness. Sections were then stained with hematoxylin and eosin (H & E) and examined for tissue damage. Colon sections were evaluated using pathology scores for evidence of inflammatory damage, as described previously [27]. 2.7. Antibodies The following fluorescence-conjugated antibodies (Abs) were used: CD4 (GK1.5), CD8α (536-7), CD11c (N418), CD86 (GL-1), CD103 (2E7), F4/80 (BM8), MHC class II (M5/114.15.2), Ly-6G (1A8), NK1.1 (PK136), TCR-β (H57-597), TCR-γδ (GL3), anti-IFN-γ (XMG1.2), and anti-IL-17 (TCC11-18H10.1). All antibodies were obtained from BioLegend (San Diego, CA, USA). 2.8. Preparation of Lamina Propria (LP) Single Cell Suspensions The LP single cell suspensions were prepared as described in detail previously [27]. In brief, colons were isolated and washed twice in ice-cold PBS. The tissues were cut open longitudinally, and mucus and gross debris were quickly removed by covering the specimen with dry paper towels. The samples were cut into 0.5–1 cm pieces. Intestinal epithelial cells (IECs) were separated from intestinal pieces by incubating in 0.15% DTT-HBSS (both from Sigma-Aldrich) buffer, with shaking, for 30 min at 37 ˝ C.
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The released IECs were removed by filtration through a mesh screen. After epithelial cell removal, the LP cells were collected by mincing the remaining tissue into 1 to 2 mm pieces, followed by digestion with 2% FBS containing collagenase (Sigma-Aldrich), with shaking, for 30 min at 37 ˝ C. The cells were filtered through a 100 µm nylon mesh, washed, and the pellet was re-suspended in RPMI-1640 and layered over 1.077 Histopaque (Sigma-Aldrich). After centrifugation at 1700ˆ g for 10 min, the light density fraction (
