Preventive effects of Goji berry on dextran-sulfate ...

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ScienceDirect Journal of Nutritional Biochemistry 40 (2016) 70 – 76

Preventive effects of Goji berry on dextran-sulfate-sodium-induced colitis in mice Yifei Kang a , Yansong Xue a , Min Du b, Mei-Jun Zhu a,⁎ b

a School of Food Science, Washington State University, Pullman, WA, 99164, USA Department of Animal Science, Washington State University, Pullman, WA, 99164, USA

Received 19 May 2016; received in revised form 28 September 2016; accepted 21 October 2016

Abstract Goji berry (Lycium barbarum) exerts immune modulation and suppresses inflammation in vitro and in vivo. We hypothesized that Goji berry had beneficial effects on dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice through suppressing inflammation. Six-week-old male C57BL/6 mice were supplemented with a standard AIN-93G diet with or without 1% (w/w) Goji berry for 4 weeks. Then, colitis was induced by supplementing 3% DSS in drinking water for 7 days, followed by 7 days of remission period to mimic ulcerative colitis symptoms. Goji berry supplementation ameliorated DSS-induced body weight loss, diminished diarrhea and gross bleeding, and resulted in a significantly decreased disease activity index, as well as DSS-associated colon shortening. Moreover, 30% mortality rate caused by DSSinduced colitis was avoided because of Goji berry supplementation. Histologically, Goji berry ameliorated colonic edema, mucosal damage and neutrophil infiltration into colonic intestinal tissue in response to DSS challenge, which was associated with decreased expression of chemokine (C-X-C motif) ligand 1 and monocyte chemoattractant protein-1, as well as inflammatory mediators interleukin-6 and cyclooxygenase-2. In conclusion, Goji supplementation confers protective effects against DSSinduced colitis, which is associated with decreased neutrophil infiltration and suppressed inflammation. Thus, dietary Goji is likely beneficial to inflammatory bowel disease patients as a complementary therapeutic strategy. Published by Elsevier Inc. Keywords: DSS; Goji berry; Gut; Epithelium; Inflammation; Inflammatory bowel disease

1. Introduction Inflammatory bowel diseases (IBDs) encompass two major forms, ulcerative colitis (UC) and Crohn's disease (CD), which are chronic intestinal autoimmune diseases characterized by diarrhea, abdominal pain and rectal bleeding. IBD incidence has increased rapidly in the past few decades in both industrialized countries and many developing countries [1]. As a chronic disease, IBD consists of relapse and remission episodes. The major goal of therapeutic treatments is to reduce relapse and maintain the remission of symptoms [2]. These treatments include anti-inflammatory or immunosuppressive drugs, such as 5-aminoaslicylic acid, 6-mercaptopurine and antibiotics. However, side effects such as fever, cramps, diabetes and high blood pressure limit their long-

Abbreviations: CD, Crohn's disease; COX-2, cyclooxygenase-2; CXCL-1, chemokine (C-X-C motif) ligand 1; DAI, disease activity index; DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon gamma; IL, interleukin; LBP, Lycium barbarum polysaccharide; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metallopeptidase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; qRT-PCR, quantitative reverse transcriptase PCR; TNBS, 2,4,6trinitrobenzene sulfonic acid; TNF-α, tumor necrosis factor alpha; UC, ulcerative colitis.; VAAM-1, vascular cell adhesion molecule ⁎ Corresponding author at: School of Food Science, Washington State University, Pullman, WA, 99164, USA. Tel.: +1 509 335 4016; fax: +1 509 335 4815. E-mail address: [email protected] (M.J. Zhu). http://dx.doi.org/10.1016/j.jnutbio.2016.10.009 0955-2863/Published by Elsevier Inc.

term use [3]. Alternative strategies for IBD management without causing undesirable side effects are needed. Accumulating evidence suggests that certain dietary supplementations can confer protective effects against IBD [4]. For instance, grape seed extract improves epithelial structure and barrier function, and suppresses inflammation in immune-deficient interleukin (IL)-10 knock out mice to reduce the risk of IBD [5,6]. Dietary supplementation of black raspberry [7] and blueberry [8] exhibits anti-inflammatory effects on dextran sulfate sodium (DSS)-induced colitis. A clinical study with a large cohort of patients showed that long-term consumption of dietary fibers reduces the risk of IBD, among which fiber derived from fruits confers the strongest effect [9]. Goji berry, fruit of Lycium barbarum, is a traditional Chinese tonic food and exerts health-beneficial properties [10]. The L. barbarum polysaccharide (LBP) containing extract exhibits neuroprotective effects in different models of Alzheimer diseases, including betaamyloid peptide neurotoxicity [11], homocysteine-induced neuronal damage and glutamate excitotoxicity [12], which are attributed to the reduced apoptosis and necrosis in cortical neurons. Recently, a milkbased Goji berry preparation named lacto-wolfberry, which contains 50% Goji berry and 25% skimmed milk, was shown to increase mice resistance to influenza infection through enhancing T-cell proliferation [13] and to exert protective roles in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice through anti-inflammatory effects [14]. However, its efficacies in different colitis models and in different colitis stages are unknown. Neither is there information on the

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percent area positive as extent (0, none; 1, b25%; 2, 25%–50%; 3, 50%–75%; 4, N75%) and depth (0, none; 1, b1/3; 2, 1/3–2/3; 3, N2/3) of infiltration.

beneficial effects of dietary Goji berry alone on intestinal inflammation, structure and overall health under inflammatory conditions. DSS-induced colitis model is one of the most frequently used IBD models, which provides high uniformity and reproducibility of lesions mostly in the distal colon, mimicking human UC symptoms morphologically and pathophysiologically [15]. This model features weight loss, rectal bleeding and diarrhea associated with colon shortening, barrier dysfunction, inflammation and immune cell infiltration [16]. This study aims to investigate the protective effects of dietary Goji berry on IBD symptoms using a DSS-induced colitis mouse model.

The total RNA was extracted from colonic tissues by TRIzol Reagent (Sigma, St. Louis, MO, USA) followed by purification using RNeasy MiniRNA kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, UsA). qRT-PCR was conducted on a Bio-Rad CFX96 real-time thermocycler using β-tubulin as the housekeeping gene as previously described [22]. SYBR Green Master Mix (Bio-Rad) was used for all PCRs. Primers sequences were listed in Table S1.

2. Materials and methods

2.6. Immunoblotting analyses

2.1. Animal care and experimental design

Immunoblotting analyses were conducted as previously described [19,23]. The band density of target protein was normalized to the β-tubulin. Antibodies against IL-6 and cyclooxygenase-2 (COX-2) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-tubulin antibody was purchased from Sigma (St. Louis, MO, USA). Horseradish-peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Cell Signaling Technology.

2.1.1. Mice and dietary treatments C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and bred in the Experimental Animal Laboratory Unit at Washington State University. All animal procedures were approved by the Washington State University Animal Care and Use Committee (ASAF#04316). Male mice were housed in a temperature-controlled room with 12:12-h light–dark cycle and fed a standard rodent diet (2018 Teklad Global 18% Protein Rodent Diet, Harlan Laboratory, Madison, WI, USA) and tap water ad libitum until dietary trial. At age of 6 weeks, 29 mice were randomly assigned to 2 groups receiving either a control diet (n=15, AIN-93G purified diet, Harlan Laboratory) or a diet supplemented with Goji berry (n=14, 1% of dry feed weight, USDA-certified organic Goji berry powder, Live Superfoods, Bend, OR, USA) for 4 weeks, when mice were challenged with 3% (w/v) DSS in water to induce colitis. Feed intake and body weight were monitored weekly during dietary treatments and DSS treatment. 2.1.2. Induction of colitis Four weeks post dietary supplementation, mice in each dietary group were randomly divided into two subgroups receiving 0 (Con, n=5; Goji, n=5) or 3% (w/v) DSS (Millipore, Billerica, MA, USA) for 7 days to induce colitis (DSS-Con, n=10; DSS-Goji, n=9), followed by plain drinking water for 7 days to recover (Fig. S1). All mice were under their respective diets during DSS induction and recovery and had free access to water and food. 2.1.3. Assessment of symptoms and colitis score The disease activity index (DAI) was calculated based on clinical symptoms of colitis including body weight, stool consistency, fecal bleeding and diarrhea, which were recorded daily [17]. 2.2. Tissue collection At necropsy, mice were anesthetized intraperitoneally with tribromoethanol (250 mg/kg body weight) followed by cervical dislocation. Blood was collected by cardiac puncture, and the resulting serum was stored at −80°C until assayed. The colon section was dissected, and the length from proximal to distal was further measured. A 5-mm segment of the distal colon was fixed in freshly prepared 4% (w/v) paraformaldehyde (pH 7.0), processed and embedded in paraffin. The remaining colon tissue was frozen in liquid nitrogen and stored at −80°C for further biochemical analyses. 2.3. Histological examination Paraffin-embedded colonic gut tissues were sectioned at 5-μm thickness and subjected to hematoxylin–eosin (H&E) staining. Each colonic section was scored as previously described [18] with modifications. Briefly, the score system was based on three independent parameters: severity of inflammation (0–3), depth of injury (0–3) and crypt damage (0–4). The summation of these scores provides a total pathobiological score with 0 being a normal tissue and 10 being the most extensive/severe disease symptoms. Mice that died during DSS induction and recovery period were assigned a score of 10, which were excluded from any other analysis. Three sections per animal that covered the length of 600 μm of the distal colon were stained and evaluated in a blinded manner by two independent researchers. 2.4. Immunohistochemical staining of neutrophils Immunohistochemical staining was conducted as previously described [19]. Briefly, colonic tissue sections were deparaffinized, rehydrated, antigen retrieved and blocked for 30 min with 1.5% goat normal serum and incubated with anti-Ly-6B.2 mAb (AbD Serotec, Raleigh, NC, USA) overnight at 4°C. Sections were then washed with phosphatebuffered saline with 0.05% Tween 20 and incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min. Signal was visualized using Vectastain ABC and DAB kits (Vector Laboratories). After counterstaining with hematoxylin, images were taken using Lecia DM2000 LED light microscope (200×, Leica Microsystems Inc., Chicago, IL, USA). Neutrophil infiltration was scored semiquantitatively from 0 (normal tissue) to 7 (intensive staining) based upon the depth (0–3) and extent of infiltration (0–4) [20,21] by two trained examiners in a blind manner. Three sections per animal were used for measurement, and for each section, a total of three to five areas encompassing N90% of the total mucosa were analyzed to determine the

2.5. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis

2.7. Statistical analysis Data were analyzed as a complete randomized design by GLM (2000); group means were expressed as mean ± S.E.M. A significant difference was considered as P value ≤.05.

3. Results 3.1. Dietary Goji berry treatment ameliorated disease activity in DSSinduced colitis Goji berry supplementation had no effect on average daily feed intake and body weight gain (Fig. S1). DSS treatment resulted in dramatic body weight loss, watery and bloody diarrhea and even mortality in mice (Fig. 1); however, dietary Goji supplementation ameliorated body weight loss during DSS induction and recovery phase (Fig. 1A). Three mice in DSStreated Con group died during the DSS induction, but no mice were lost in DSS-Goji group (Fig. 1B). Consistently, the severity of disease symptoms as indicated by DAI score was mitigated in Goji-berry-supplemented mice during DSS induction and recovery phase (Fig. 1C). As expected, without DSS induction, mice in both Con and Goji group showed no symptom (Fig. 1C). In line with improved body weight loss and DAI score, colon length shortening, an indirect marker of inflammation [24], was improved in DSS group supplemented with Goji berry compared with mice treated with DSS alone (Fig. 1D and E). 3.2. Dietary Goji berry alleviated DSS-induced mucosal damage DSS treatment induced colonic structure damages, which were characterized by severe lesions throughout the mucosa, alteration of crypt structure, enhanced infiltration of immune cells into the mucosal and submucosal layer, and colonic edema (Fig. 2A). In mice supplemented with Goji berry, the histological damages induced by DSS treatment were substantially less (Fig. 2A), which were associated with reduced pathobiological score (Fig. 2B). Accompanied with damaged intestinal structure, DSS treatment resulted in increased pore-forming tight junction protein Claudin 2 and decreased barrier-forming tight junction protein ZO-2 (Fig. 3A and B), as well as up-regulated expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) (Fig. 3C). These alterations were numerically restored by Goji berry supplementation, although they were not statistically different (Fig. 3). 3.3. Dietary Goji berry suppressed inflammation in the colonic tissues Based on immunohistochemical staining against Ly-6B, which recognizes neutrophils and a portion of activated macrophages [25], DSS treatment induced extensive and severe neutrophil infiltration in

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Fig. 1. Symptoms of DSS-induced colitis were ameliorated with dietary Goji supplement. (A) Percentage of initial body weight. (B) Survival rate. (C) DAI; score 0 represents no disease symptoms, and score 12 represents the most severe symptoms. (D) Colon length at necropsy. (E) Representative images of colon length. Data indicate mean ± S.E.M., n=9–10 and 5 for mice with and without DSS induction, respectively. #P≤.1, *P≤.05, **P≤.01, *** P≤.001.

colonic tissue (Fig. 4). Goji berry supplementation mitigated neutrophil infiltration into colonic mucosa in DSS-treated mice, while no neutrophil infiltration was observed in mice without DSS treatment regardless of dietary treatments (Fig. 4). In line with severe neutrophil infiltration, mRNA expressions of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-1β, IL-6, the inducible nitric oxide synthase (iNOS) and inflammatory enzyme COX-2 were markedly increased in the DSS-treated group (Fig. 5). Furthermore, IL-6 and COX-2 protein contents were enhanced in the respective DSS-treated

groups (Fig. 5A and B). Of note, Goji berry supplementation mitigated the up-regulation of IL-6 and COX-2 mRNA expression and protein content in DSS-treated mice (Fig. 5A and B). 3.4. Dietary Goji berry suppresses neutrophil infiltration through inhibiting chemoattractant molecules The intestinal chemokines are critical mediators in neutrophil recruitment and infiltration. The IL-8 is a potent chemoattractant for


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Fig. 2. Dietary Goji supplementation ameliorated DSS-induced histological alteration. (A) Representative images of H&E staining taken under 200×. (B) Pathological score (0–10), calculated by the sum of crypt damage (0–4), severity of inflammation (0–3) and depth of injury (0–3), with 0 being a normal tissue and 10 exhibiting the most extensive/severe disease symptoms. Each symbol represents one animal. Mice that died during the DSS treatment was assigned the score of 10; n=9–10 and 5 for mice with and without DSS induction, respectively. Data indicate mean ± S.E.M. *P≤.05, ***P≤.001.

neutrophils in human [26,27] and has a central role in mediating inflammation in colitis. The chemokine (C-X-C motif) ligand 1 (CXCL1) has a similar function as IL-8 in recruiting and activating murine neutrophils [28]. Consistent with enhanced neutrophil infiltration, CXCL-1 was enhanced in DSS-treated mice regardless of dietary treatments. Goji berry supplementation alleviated the up-regulation of CXCL-1 (Fig. 6A). Additionally, the expression of monocyte chemoattractant protein-1 (MCP-1), another key chemokine, was markedly enhanced in DSS-treated mice, which was alleviated by Goji berry consumption (Fig. 6A). Neutrophils produce matrix metallopeptidases (MMPs) that have critical roles in recruitment of additional effector cells to the inflamed tissue. Accordingly, MMP-9 was markedly enhanced in response to DSS induction, which was numerically ameliorated by Goji berry supplementation (Fig. 6B).

4. Discussion 4.1. Goji berry supplementation reduced mortality and symptoms of DSS-induced colitis Goji berry and its extracts are known for their beneficial effects on prevention of various chronic diseases, including glaucoma, Alzheimer's disease, diabetes and fatty liver diseases [29]. However, the knowledge about impacts of Goji berry on intestinal health is limited. This study showed that dietary Goji berry had protective effects against DSSinduced colitis as indicated by reduced mortality in response to DSS treatment, as well as by reduced body weight loss, overall DAI and colon length shortening among the remaining survivors. Such protective effect of Goji berry was likely due to its high polysaccharide content. LBP is the most well-recognized and important group of phytonutrient in Goji berry, which typically accounts for 5%–8% of the dried fruit depending on the source of Goji berry and extraction method [30]. In support of our findings, Goji berry extract, LBP, exerted protective effects on intestinal injury in ischemia–reperfusion-induced intestinal damage in rat model, likely through suppressing oxidative stress and inflammation [31]. Goji and milk product lacto-wolfberry reduced weight loss and inflammation in TNBS-induced acute colitis where mice

were subjected to treatment with TNBS (125 mg/kg in 50% ethanol) solution for 4 days and then sacrificed [14]. 4.2. Dietary Goji berry alleviated DSS-induced mucosal damage Consistent with previous reports [32], DSS treatment in C57/B6 mice induced colonic epithelial structural damage. Goji supplementation alleviated disease symptoms and colonic lesions incurred by DSS in survival mice, clearly showing the effectiveness of a low-dose Goji supplementation in improving IBD symptoms. IBD is associated with abnormal expression of junction complex proteins and impaired intestinal barrier integrity. Consistently, DSS treatment resulted in alteration of tight junction protein contents. These alterations in mice supplemented with Goji berry were numerically restored compared to control mice surviving DSS treatment. The reasons for lacking of significant difference could be due to the death of three mice in the control group, which resulted in the loss of mice with the severest symptoms and also smaller sample size. Consistently, in cultured retinal pigment epithelial cells, addition of Goji extract was able to restore the transepithelial electrical resistance impaired by high glucose [33]. In ischemia–reperfusion injury rat model, the impaired intestinal permeability was restored by LBP supplementation [31]. 4.3. Goji berry supplementation suppressed inflammation and neutrophil infiltration in DSS-induced colitis Neutrophils play a crucial role in the intestinal homeostasis. Nevertheless, under IBD condition, excessive recruitment and activation of neutrophil in intestine result in mucosal injury and exaggerated symptoms. As a result, the inhibition of neutrophil infiltration confers protective effects on the intestine. In line with the reduced disease symptoms and improved pathobiological damage, Goji berry supplementation resulted in markedly reduced neutrophil infiltration in DSStreated mice. In support of our finding, in the TNBS-induced colitis model, lacto-wolfberry confers protective effects associated with reduced colonic myeloperoxidase level, a marker of neutrophils [14].

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Fig. 3. Tight junction protein and adhesion molecules in mice under dietary and/or DSS treatment. (A and B) Representative images and statistical data of tight junction protein Western blotting. (C) mRNA expression of adhesion molecules. Data indicate mean ± S.E.M., n=7–9 and 5 for mice with and without DSS induction, respectively. *P≤.05, **P≤.01.

The overproduction of inflammatory mediators including cytokines and chemokines fuels inflammatory cascade reactions and therefore colon damage in IBD [34]. Proinflammatory cytokines play important roles in the recruitment of immune cells [35]. In the inflamed colon, the up-regulation of IL-1β and TNF-α is involved in inflammatory leukocyte recruitment and retention [36]. IL-6, a key mediator of chronic inflammation of UC, is produced by T cells and macrophages in the inflamed mucosa [37]; suppression of IL-6 production was considered as a target for therapeutic treatment of IBD [38]. In addition, COX-2 is suppressed under normal cellular condition but is elevated in response to inflammation [39]. The expression of COX-2 is up-regulated in UC [40] and colorectal cancer [41]. The nonsteroidal anti-inflammatory drugs, which inhibit COX-2, confer protective effect in IBD [42]. Therefore, the COX-2 silencing strategy by engineered bacterial strain was an effective and safe tool for the therapy of IBD [43]. As a result, the inhibitory effects of Goji berry on neutrophil infiltration as well as IL-6 and COX-2 production in DSS-induced colitis confer its great potential to be used as a complementary therapeutic approach for alleviating IBD symptoms as well as preventing colitis-associated colorectal cancer.

Fig. 4. Neutrophil immunohistochemical staining. (A) Scoring criteria of neutrophil infiltration. The infiltration of neutrophil was scored semiquantitatively from 0 (normal tissue) to 7 (intensive staining) based upon the depth and extent of infiltration; images magnified at 400×. (B) Neutrophil scores. Each symbol represented one animal. Data indicate mean ± S.E.M., *P≤.05, ***P≤.001.

The intestinal chemokine molecules play critical roles in neutrophil infiltration. CXCL-1, previously called KC/GROα, is a member of CXC chemokine family and one of the potent chemoattractants that contributes to the recruitment and activation of neutrophils in inflamed tissues [44]. CXCL-1 was induced significantly as colitis developed [28]. MCP-1, a member of CC chemokines, functions to attract monocytes, macrophages and lymphocytes [45], further eliciting inflammation in the colon of IBD. In agreement with their functions and improved neutrophil infiltration and pathobiological scores, both CXCL-1 and MCP-1 expressions were decreased in DSS-treated mice supplemented with Goji berry. Our data are consistent with a previous report showing


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Fig. 5. Inflammatory markers in mice under dietary and/or DSS treatment. (A) IL-6 mRNA (top) and protein (bottom) content. (B) COX2 mRNA (top) and protein (bottom) content. (C) mRNA expression of IL-1β, IFN-γ and TNF-α. (D) iNOS mRNA expression. Data indicate mean ± S.E.M., respectively. *P≤.05, **P≤.01.

the positive correlation of MCP-1 expression with inflammation in colon tissue [46]. Additionally, MMPs have critical roles in recruitment of additional effector cells to the inflamed tissue through cleavage of chemokine precursors. Overexpression of MMP-9 in intestinal epithelium is associated with increased levels of CXCL-1 mRNA [47]. Accordingly, MMP-9 was markedly enhanced in DSS-treated mice, which was numerically ameliorated by Goji berry supplementation. Taken together, dietary Goji berry treatment alleviated mucosal damage, which was

associated with down-regulation of chemoattractant expression and reduced immune cell infiltration. In conclusion, Goji berry supplementation protects mice against DSSinduced colitis and mucosal injury, which is associated with suppression of neutrophil infiltration and expression of chemoattractant molecules CXCL-1 and MCP-1, as well as decreased proinflammatory mediators IL-6 and COX-2. Altogether, our data suggested that dietary Goji berry has the potential to be used as a complementary therapeutic approach for alleviating IBD symptoms.

Fig. 6. mRNA expression of chemoattractant molecules in mice under dietary and/or DSS treatment. (A) CXCL-1 and MCP-1 mRNA expression. (B) MMP-9 mRNA expression. Data indicate mean ± S.E.M., *P≤.05, **P≤.01.

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Acknowledgment This study is partially funded by Emerging Research Issues Internal Competitive Grant from the Washington State University, College of Agricultural, Human, and Natural Resource Sciences, Agricultural Research Center project 10A-3057-8640. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.jnutbio.2016.10.009. References [1] Loftus CG, Loftus Jr EV, Harmsen WS, Zinsmeister AR, Tremaine WJ, Melton III LJ, et al. Update on the incidence and prevalence of Crohn's disease and ulcerative colitis in Olmsted County, Minnesota, 1940–2000. Inflamm Bowel Dis 2007;13:254–61. [2] Alrubaiy L, Rikaby I, Sageer M, Hutchings HA, Williams JG. Systematic review of the clinical disease severity indices for inflammatory bowel disease. Inflamm Bowel Dis 2015;21:2460–6. [3] Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 2007;369:1641–57. [4] Durchschein F, Petritsch W, Hammer HF. Diet therapy for inflammatory bowel diseases: the established and the new. World J Gastroenterol 2016;22:2179–94. [5] Yang G, Wang H, Kang YF, Zhu MJ. Grape seed extract improves epithelial structure and suppresses inflammation in ileum of IL-10-deficient mice. Food Funct 2014;5:2558–63. [6] Yang G, Xue Y, Zhang H, Du M, Zhu MJ. Favourable effects of grape seed extract on intestinal epithelial differentiation and barrier function in IL10-deficient mice. Br J Nutr 2015;114:15–23. [7] Montrose DC, Horelik NA, Madigan JP, Stoner GD, Wang LS, Bruno RS, et al. Antiinflammatory effects of freeze-dried black raspberry powder in ulcerative colitis. Carcinogenesis 2011;32:343–50. [8] Pervin M, Hasnat MA, Lim JH, Lee YM, Kim EO, Um BH, et al. Preventive and therapeutic effects of blueberry (Vaccinium corymbosum) extract against DSS-induced ulcerative colitis by regulation of antioxidant and inflammatory mediators. J Nutr Biochem 2016;28:103–13. [9] Ananthakrishnan AN, Khalili H, Konijeti GG, Higuchi LM, de Silva P, Korzenik JR, et al. A prospective study of long-term intake of dietary fiber and risk of Crohn's disease and ulcerative colitis. Gastroenterology 2013;145:970–7. [10] Amagase H, Farnsworth NR. A review of botanical characteristics, phytochemistry, clinical relevance in efficacy and safety of Lycium barbarum fruit (goji). Food Res Int 2011;44:1702–17. [11] Yu MS, Leung SKY, Lai SW, Che CM, Zee SY, So KF, et al. Neuroprotective effects of anti-aging oriental medicine Lycium barbarum against beta-amyloid peptide neurotoxicity. Exp Gerontol 2005;40:716–27. [12] Ho YS, Yu MS, Yik SY, So KF, Yuen WH, Chang RC. Polysaccharides from wolfberry antagonizes glutamate excitotoxicity in rat cortical neurons. Cell Mol Neurobiol 2009;29:1233–44. [13] Ren Z, Na L, Xu Y, Rozati M, Wang J, Xu J, et al. Dietary supplementation with lactowolfberry enhances the immune response and reduces pathogenesis to influenza infection in mice. J Nutr 2012;142:1596–602. [14] Philippe D, Brahmbhatt V, Foata F, Saudan Y, Serrant P, Blum S, et al. Antiinflammatory effects of lacto-wolfberry in a mouse model of experimental colitis. World J Gastroenterol 2012;18:5351–9. [15] Valatas V, Vakas M, Kolios G. The value of experimental models of colitis in predicting efficacy of biological therapies for inflammatory bowel diseases. Am J Physiol Gastrointest Liver Physiol 2013;305:G763–85. [16] Yan Y, Kolachala V, Dalmasso G, Nguyen H, Laroui H, Sitaraman SV, et al. Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis. PLoS One 2009;4, e6073. [17] Hamamoto N, Maemura K, Hirata I, Murano M, Sasaki S, Katsu K. Inhibition of dextran sulphate sodium (DSS)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules (endothelial leucocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1)). Clin Exp Immunol 1999; 117:462–8. [18] Schepp-Berglind J, Atkinson C, Elvington M, Qiao F, Mannon P, Tomlinson S. Complement-dependent injury and protection in a murine model of acute dextran sulfate sodium-induced colitis. J Immunol 2012;188:6309–18. [19] Zhu MJ, Du M, Hess BW, Nathanielsz PW, Ford SP. Periconceptional nutrient restriction in the ewe alters MAPK/ERK1/2 and PI3K/Akt growth signaling pathways and vascularity in the placentome. Placenta 2007;28:1192–9. [20] Fournier BM, Parkos CA. The role of neutrophils during intestinal inflammation. Mucosal Immunol 2012;5:354–66.

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