Oral Administration of Hen Egg White Ovotransferrin Attenuates the

Jan 20, 2015 - Ovotransferrin (OVT), one of the major hen egg white proteins, was shown .... Effects of OVT on the Development of DSS-Induced Colitis ...
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Oral Administration of Hen Egg White Ovotransferrin Attenuates the Development of Colitis Induced by Dextran Sodium Sulfate in Mice Yutaro Kobayashi,†,# Prithy Rupa,† Jennifer Kovacs-Nolan,† Patricia V. Turner,§ Toshiro Matsui,# and Yoshinori Mine*,† †

Department of Food Science and §Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada Division of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

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ABSTRACT: Ovotransferrin (OVT), one of the major hen egg white proteins, was shown to possess antimicrobial and antioxidant activities in vitro. However, there is no information regarding the in vivo preventative effect in chronic inflammatory diseases such as inflammatory bowel disease (IBD). The aim of the present study is to evaluate the anti-inflammatory effects of OVT in a mouse model of dextran sodium sulfate (DSS)-induced colitis. OVT (50 or 250 mg/kg BW) was given orally for 14 days to female BALB/c mice, and 5% DSS (MW 36−50 kDa) was used to induce acute colitis (days 7−14) via drinking water. The current in vivo study demonstrated that OVT significantly reduced clinical signs, weight loss, shortening of the colon, and inflammatory cytokine markers of disease. The histopathological analysis of the colon revealed that OVT reduced histological scores. These results indicate that the use of OVT may be a potential promising candidate for the prevention of IBD. KEYWORDS: ovotransferrin, egg white protein, inflammatory bowel disease, dextran sodium sulfate, inflammatory cytokine

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INTRODUCTION Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic inflammation on the gastrointestinal tract. Both UC and CD have similar profiles in the colon, which include diarrhea, rectal bleeding, abdominal pain, and weight loss. CD involves transmural inflammation with inflammatory processes occurring in the gastrointestinal tissue, whereas UC is characterized by limited mucosal inflammation on the colon. The pathogenesis of IBD is not completely understood, but it is believed to be developed by complex interactions of multiple immunological factors that includes genetic, environmental, and microbiological components that play a significant role. The imbalance in the mucosal immune response leads to overproduction of inflammatory cytokines, chemokines, oxidants, and matrix metalloproteinase, resulting in prolonged inflammatory responses and irreparable tissue damage.1 Clinical studies have shown that the risk of colorectal cancer in patients with UC is increased by several factors, such as prolonged oxidative stress and immune response.2 So far, a number of immunosuppressive drug and antibody-related treatment strategies3 have been developed for curing IBD and targeting specific signaling pathways/pro-inflammatory cytokines. Due to adverse side effects of these drugs with long-term use,4 there is a growing interest in alternative treatment regimens that are beneficial and safe, such as dietary interventions. Recent studies have demonstrated that some dietary compounds, including dietary egg lysozyme,5 egg yolk peptides,6−9 soy peptides,10,11 amino acids,12,13 mannanase-hydrolyzed copra meal,14 and plant phytochemicals,15,16 can exhibit anti-inflammatory and antioxidant properties to potentially ameliorate the severity of IBD. It has been well reported that hen egg and its components play a beneficial role in the prevention and treatment of chronic © 2015 American Chemical Society

infectious diseases. Egg white proteins, such as ovalbumin, ovotransferrin (OVT), and lysozyme, and egg york phosvitin possess antibacterial activity and antioxidant activity.17 It was earlier demonstrated that ovalbumin glyconjugate had immunomodulating activities in mice.18 The anti-inflammatory effect of lysozyme was shown in a porcine model of dextran sodium sulfate (DSS)-induced colitis.5 OVT, constituting about 12% of egg white proteins,17 is an iron-binding glycoprotein that is composed of 686 amino acids. OVT and its hydrolysates have been reported to have protective effects against oxidative stress-induced DNA damage in leukocytes.19 However, the preventative effects of OVT in chronic inflammation-related diseases such as IBD are yet to be elucidated. The present study was undertaken to investigate antiinflammatory effects of OVT against DSS-induced colitis in mice and elucidate potential mechanisms whereby OVT may act to improve pathophysiology of IBD. OVT (50 or 250 mg/ kg BW) was given orally for 14 days to female BALB/c mice, and 5% DSS (MW 36−50 kDa) was used to induce acute colitis (days 7−14) via drinking water.

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MATERIALS AND METHODS

Preparations for Ovotransferrin. The crude OVT was obtained from Sigma-Aldrich (St. Loius, MO, USA) and purified using a MonoQ5 column (10 × 64 mm, Bio-Rad Laboratories, Hercules, CA, USA). Briefly, the crude OVT was dissolved in 0.05 M Tris-HCl buffer (pH 7.0), and 50 mg protein was loaded onto the column using the BioRad Biologic HPLC system (Bio-Rad). The protein was eluted by a linear gradient of 0−0.5 M Tris-HCl buffer (pH 7.0) at a flow rate of Received: Revised: Accepted: Published: 1532

October 30, 2014 January 15, 2015 January 20, 2015 January 20, 2015 DOI: 10.1021/jf505248n J. Agric. Food Chem. 2015, 63, 1532−1539

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Journal of Agricultural and Food Chemistry

Figure 1. OVT ameliorates DSS-induced colitis. Mice were given 50 or 250 mg/kg BW OVT for 14 days. On day 7, colitis was induced with 5% DSS water. (A) Mice were weighed daily, and changes in body weight are reported as percent change relative to the initial weight. (B) Mice were scored daily for clinical signs of DSS-induced colitis. (C) After 14 days, the extracted colons were measured from the end of cecum to the anus. Data represent the mean ± SEM of n = 10/group. Unless indicated, no significance difference was observed between groups. Values without a common letter are significantly different at P < 0.05. 1.0 mL/min for 40 min. The purified OVT was dialyzed (MWCO 8000 tubing) against Milli-Q water and lyophilized. The purity was determined by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed as >90% (data not shown). Induction of Colitis and Sample Treatment. All animal experiments were approved by the University of Guelph Animal Care Committee and carried out in accordance with the Canadian Council of Animal Care Guide to the Care and Use of Experimental Animals (eAUP no. 1536). The experiments for induction of DSSinduced colitis in mice were performed as previously described.11 Briefly, 40 female BALB/c mice (6−8 weeks; Charles River Laboratories, Montreal, QC, Canada) were randomly divided into four groups (n = 10/group) and housed with a 12 h light/dark cycle with access to standard food and water ad libitum. OVT (50 or 250 mg/kg BW/day) was given by oral gavage for 14 days. On the seventh day, 5% (w/v) DSS (MW 36−50 kDa; MP Biomedical, Solon, OH,

USA) was added to drinking water to induce acute colitis for 7 days. The DSS solution was replaced every other day. The negative control group received water only. The positive group received water with DSS for 7 days. Body weights were measured on a daily basis. The daily weight changes were calculated by percent in relation to the initial weight measured. After 14 days, mice were humanely euthanized, and colons were collected and sectioned longitudinally. Colon samples were flash frozen and stored at −80 °C until further analysis. Clinical Evaluation of Colitis. Mice were examined daily for stool consistency, presence of blood in stool, and general appearance. The clinical scoring was performed as described earlier by Maxwell et al.20 and defined as follows: stool score, 0 = normal, 1 = moist/sticky stool, 2 = soft stool, 3 = diarrhea; stool blood score, 0 = no blood, 1 = evidence of blood in stool or around anus, 2 = severe bleeding; mouse 1533

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Results were expressed as fold changes relative to the negative control samples. Histological Analysis. A section of the distal colon was fixed in 10% (w/v) buffered formalin (Fisher Scientific). Paraffin-embedded sections were stained with hematoxylin and eosin (H&E) (Animal Health Laboratory, University of Guelph, Guelph, ON, Canada). The stained samples were examined using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). Histological scoring of the colitic lesions was evaluated in a double-blinded fashion by a pathologist using the previously reported scoring system11 with respect to the degree, extent, and depth of inflammation and the severity of damage of the glands and epitheliums. The total sum of histologic score ranged from 0 to 19. Statistical Analysis. The results were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad, San Diego, CA, USA). Statistical differences between curves were analyzed using two-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test. Statistical differences between individual group were determined using one-way ANOVA followed by Tukey’s multiple-comparison test. P < 0.05 was considered statistically significant.

appearance, 0 = normal, 1 = ruffled fur or altered gait, 2 = lethargic or moribund. Cytokine Concentration in Colon Samples. The colons were excised and homogenized in ice-cold phosphate-buffered saline (PBS) containing proteinase inhibitors (1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 10 μg/mL pepstain A; Sigma-Aldrich) for 3 min using a homogenizer (Polytron PT 1200, Kinematica AG, Luzern, Switzerland). The supernatants were collected following centrifugation at 10000g for 10 min. The concentration of protein was determined by using a DC protein assay (Bio-Rad), according to the manufacturer’s instruction. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 cytokine concentration were determined by ELISA as described earlier.11 IL-17A was measured using an IL-17A ELISA kit (eBioscience, San Diego, CA, USA), according to the manufacturer’s protocol. The following antibodies and standard proteins for ELISA were obtained from BD Bioscience (San Jose, CA, USA): anti-mouse TNF-α, biotin anti-mouse TNF-α, recombinant mouse TNF-α, anti-mouse IL-6, biotin anti-mouse IL-6, recombinant mouse IL-6, and avidin−horseradish peroxidase (AvHRP). Briefly, 96-well ELISA plates (Corning, Corning, NY, USA) were coated with anti-mouse TNF-α antibody (1:500, diluted in 0.2 M sodium phosphate buffer, pH 6.5) or anti-mouse IL-6 antibody (1:250, diluted in 0.1 M sodium phosphate buffer, pH 9.0) overnight at 4 °C. The plate was blocked with PBS containing 1% bovine serum albumin (BSA; Fisher Scientific, Ottawa, ON, Canada) for 1 h at 37 °C. Colon supernatant samples and diluted standards were added to the plate and incubated for 2 h at 37 °C. Biotin anti-mouse TNF-α (1:500, v/v) or anti-mouse IL-6 antibody (1:500, v/v) was further added and incubated for 1 h at 37 °C. Av-HRP (1:2000, v/v) was added for 0.5 h at 37 °C. The plate was washed three times with PBS-0.05% Tween 20 (PBS-T). Then, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma-Aldrich) was added to each well, and the reaction was stopped by adding 0.5 N H2SO4 (50 μL/well). The absorbance was measured at 450 nm by a plate reader (iMark model 550, Bio-Rad). The concentration of these cytokines was determined from the standard calibration curves and reported as picograms of cytokine relative to protein concentration in the supernatant. RNA Isolation, cDNA Synthesis, and Real-Time PCR. Total RNA from the colon was extracted according to a previous study.11 Briefly, the colon was homogenized in Trizol reagent (Life Technologies, Carlsbad, CA, USA) and total RNA was extracted by using an Aurum Total RNA Mini Kit (Bio-Rad) according to the manufacturer’s instructions. The quantity and quality of RNA were determined by Nano Drop ND-1000 (Thermo Scientific, Waltham, MA, USA). The cDNA was synthesized using the qScript cDNA synthesis kit (Quanta BioSciences, Gaithersburg, MD, USA), according to the manufacturer’s instructions. Real-time quantitative PCR was carried out using PerfeCTa SYBR Green Supermix (Quanta Biosciences) on a MyiQ single-color real-time PCR detection system (Bio-Rad) using the following conditions: denaturation for 15 s at 95 °C, annealing for 15 s at 58 °C, and extension for 30 s at 72 °C. The mouse primers used were as follows: β-actin, forward, 5′-GAAATCGTGCGTGACATCAAAG-3′, reverse, 5′-TGTAGTTCATGGATGCCACAG-3′; TNF-α, forward, 5′-CCCCAAAGGGATGAGAAGTT3′, reverse, 5′-CACTTGGTGGTTTGCTACGA-3′; IL-6, forward, 5′CCGGAGAGGAGACTTCACAG-3′, reverse, 5′-CAGAATTGCCATTGCACAAC-3′; IL-1β, forward, 5′-GGATGAGGACATGAGCACCT-3′, reverse, 5′-AGCTCATATGGGTCCGACAG-3′; interferon (IFN)-γ, forward, 5′-GCTCTTCCTCATGGCTGTTT-3′, reverse, 5′-GTCACCATCCTTTTGCCAGT-3′; monocyte chemoattractant protein (MCP)-1, forward, 5′-CCCAATGAGTAGGCTGGAGA-3′, reverse, 5′-TCTGGACCCATTCCTTCTTG-3′, IL-17A, forward, 5′CCAGGGAGAGCTTCATCTGT-3′, reverse, 5′-AGGAAGTCCTTGGCCTCAGT-3′; IL-10, forward, 5′-GCCTTATCGGAAATGATCCA-3′, reverse, 5′-AGGGGAGAAATCGATGACAG-3′. These primers were obtained from the University of Guelph Laboratory Services Molecular Biology Section (Guelph, ON, Canada). Relative gene expression was calculated according to the 2−ΔΔCt method21 using mouse β-actin as an endogenous control for normalization.

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RESULTS

Effects of OVT on the Development of DSS-Induced Colitis in Mice. To determine a possible dose-dependent effect of OVT, OVT was administered by oral gavage at two different doses: 50 and 250 mg/kg BW. The DSS treatment for 7 days exacerbated the progression of acute colitis characterized by weight loss, the presence of blood in stool, and diarrhea. As shown in Figure 1A, the ratio of changing body weight in the positive (DSS) group was significantly decreased in the last 2 days of DSS treatment (days 13 and 14 of the study, P < 0.001), compared with the negative control (water) group. The high-dose OVT (DSS + OVT250) group had significantly lower body weight loss than the DSS group on the sixth day of DSS treatment (day 13, P = 0.048 vs DSS) and tended to be lower on the seventh day (day 14, P = 0.064 vs DSS) (Figure 1A). In the low-dose OVT group (DSS + OVT50), the decrease in the ratio of weight change between days 12 and 13 tended to be lower than that in the DSS treatment (DSS, 1.7 ± 1.4 to −2.8 ± 1.8%; DSS + OVT50, 3.0 ± 1.1 to 0.6 ± 1.5%); however, no significant improvement of DSS-induced weight loss was observed (day 13, P = 0.256; day 14, P = 0.221 vs DSS) (Figure 1A). The average of initial weights did not differ among each group (water group, 17.3 ± 0.4 g; DSS group, 17.0 ± 0.3 g; DSS + OVT50 group, 17.1 ± 0.3 g; DSS + OVT250 group, 16.9 ± 0.3 g). The increase of clinical scores was also observed in the DSS group on the fifth day of DSS treatment (P < 0.001), compared with the control group. As shown in Figure 1B, low-dose OVT treatment significantly decreased the severity of DSS-induced clinical signs on the sixth and seventh days of DSS treatment (day 6, P < 0.001; day 7, P < 0.001 vs DSS) and high-dose treatment also reduced the signs on the fifth, sixth, and seventh days (day 5, P = 0.003; day 6, P < 0.001; day 7, P < 0.001 vs DSS). The consumption of water among groups showed no significant differences during the whole experiment, and no clinical signs were observed in mice before the start of DSS treatment (data not shown). The colon shortening induced by DSS has already been shown to be inversely linked with the severity of DSS-induced colitis in mice.22 As shown in Figure 1C, the significant shortening of the colon length was confirmed in the DSS group (water, 7.0 ± 0.3 cm; DSS, 4.6 ± 0.2 cm; P < 0.001 vs water), whereas both doses of OVT treatment remarkably improved the colon length 1534

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Journal of Agricultural and Food Chemistry (DSS + OVT50, 5.3 ± 0.1 cm, P = 0.039; DSS + OVT250, 5.3 ± 0.1 cm, P = 0.030 vs DSS). Effects of OVT on the Expression of Inflammatory Cytokines in DSS-Treated Mice. The anti-inflammatory effects of OVT in the colon were investigated by measuring protein and mRNA expression of the inflammatory cytokines in the colon. As shown in Figure 2, DSS treatment remarkably

doses of OVT significantly inhibited the DSS-induced expression of TNF-α and IL-6 (TNF-α, DSS + OVT50, 47.9 ± 9.8 pg/mg protein, P = 0.005; DSS + OVT250, 50.6 ± 12.6 pg/mg protein, P = 0.007; IL-6, DSS + OVT50, 102.0 ± 8.4 pg/mg protein, P = 0.009; DSS + OVT250, 73.5 ± 16.3 pg/mg protein, P < 0.001 vs DSS); high-dose OVT treatment also significantly decreased IL-17A expression (DSS + OVT50, 45.5 ± 8.6 pg/mg protein, P = 0.561; DSS + OVT250, 30.5 ± 3.4 pg/mg protein, P = 0.024 vs DSS). In addition, as shown in Figure 3, both doses of OVT significantly inhibited the mRNA expression of pro-inflammatory cytokines TNF-α (P < 0.001 vs DSS), IL-6 (P < 0.05 vs DSS), IL-1β (P < 0.05 vs DSS), IFN-γ (P < 0.01 vs DSS), MCP-1 (P < 0.05 vs DSS), and IL-17A (P < 0.01 vs DSS). However, the expression of IL-10, an antiinflammatory cytokine, was not affected by OVT treatment, suggesting that the anti-inflammatory action of OVT is likely not associated with its ability to inhibit the expression of antiinflammatory cytokines in the colon. Effect of OVT on the Histological and Morphological Damage in DSS-Treated Mice. The treatment effects of OVT on histological and morphological damages in the colons were examined following the histological grading system as described before.11 The observations were confirmed by estimation of biological parameters relevant to colonic inflammation. Colonic inflammation is characterized by severe lesions in the mucosa, alteration of epithelial structure, increase in neutrophil population and lymphocyte infiltration into the mucosal and submucosal areas, and loss of crypts. The colons from the negative control group displayed typical characteristic features of normal glands, abundant goblet cells in epithelium, no mucosal hyperplasia, and a small amount of infiltrated immune cells (Figure 4A). In contrast, the DSS-treated colons displayed severe epithelial damage with extensive loss of glands, depletion of the goblet cells, thickening of the mucosa, cellular infiltration into the submucosa and lamina propria, and destruction of the architecture (Figure 4B), resulting in a high histological score (Figure 4E). Both doses of OVT remarkably (P < 0.05) reduced the total histological score (Figure 4E) compared with the DSS group. As shown in Figure 4C,D, OVT-treated colons displayed a decrease in inflammatory cellular infiltration of cells with mild loss of epithelial cells and less expansion of the lesion area. These data suggested that OVT treatment improved the colonic inflammation based on histological analysis, which confirms beneficial preventative effects of OVT on colitis progression.

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DISCUSSION In the present study, we have made progress toward elucidating the mechanism of action of OVT in the amelioration of DSSinduced colitis in mice. Currently, most conventional medical treatments for IBD are mainly based on the use of antiinflammatory drugs such as 5-aminosalicylates or biological agents such as anti-TNF-α antibody. However, the long-term use of these immunosuppressive agents is associated with strong side effects, including infectious complications that weaken the immune system, resulting in malignancies and severe heart failure.4 Therefore, complementary and effective treatments for IBD are needed. Several dietary intervention trials have reported beneficial effects in maintaining remission or improving recovery of lesion in IBD patients.23−28 Our previous in vivo and in vitro studies demonstrated that some egg proteins or peptides5−9 could ameliorate the colonic inflammatory responses and the development of colitis. OVT

Figure 2. Effect of OVT on inflammatory cytokine protein expression in the colon of DSS-treated mice. Protein expression of TNF-α, IL-6, and IL-17A was determined by ELISA. Results were expressed as picograms of cytokine relative to protein concentration in the colon supernatant. Data represent the mean ± SEM of n = 10/group. Values without a common letter are significantly different at P < 0.05.

increased the protein expression of TNF-α, IL-6, and IL-17A, compared to the water group (TNF-α, water, 55.4 ± 3.4 pg/mg protein, DSS, 105.4 ± 11.8 pg/mg protein, P = 0.014; IL-6, water, 53.2 ± 6.2 pg/mg protein, DSS, 163.9 ± 14.0 pg/mg protein, P < 0.001; IL-17A, water, 26.7 ± 2.9 pg/mg protein, DSS, 55.3 ± 5.6 pg/mg protein, P = 0.010 vs water). Both 1535

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Figure 3. Effect of OVT on inflammatory cytokine gene expression in the colon of DSS-treated mice. Relative mRNA expressions of TNF-α, IL-6, IL-1β, IFN-γ, MCP-1, IL-17A, and IL-10 were measured by real-time PCR. Results were expressed as fold change relative to negative control mice. Data represent the mean ± SEM of n = 10/group. Values without a common letter are significantly different at P < 0.05.

has been shown earlier to possess antibacterial activity and antioxidant activity,17 but the anti-inflammatory effect of OVT on IBD has not been performed yet. To the best of our knowledge, the present study is the first of its kind to demonstrate that OVT (50 or 250 mg/kg BW) successfully attenuated DSS-induced intestinal inflammation in mice. In macroscopic and clinical evaluation analyses, both doses of OVT significantly inhibited clinical signs and colon shortening in DSS-induced colitis mice. In addition, OVT significantly suppressed local expression of cytokines (TNF-α, IL-6, IL-1β, IFN-γ, MCP-1, and IL-17A) in the colon. These results suggested that OVT would block the pathological progress of UC by inhibiting cytokine production. Likewise, histological analysis revealed that OVT improved the function of colon impaired by DSS induction, suggesting OVT was able to attenuate clinical symptoms of colitis in the present study, even at low dose (50 mg/kg BW). The present study demonstrated that OVT significantly decreased the clinical score and prevented the colon shortening induced by DSS treatment. In addition, the improvement of weight loss was shown in the high-dose group. Cytokines play a

crucial role in developing the perpetuating inflammation in IBD. The recruitment of macrophages, neutrophils, and T cells are enhanced by cytokines, resulting in dysregulated immune response and chronic inflammation.29 Increases in the expression of cytokines, such as TNF-α, IL-6, IL-1β, IFN-γ, and MCP-1, have been shown in both mouse models of DSSinduced colitis30,31 and IBD patients.32,33 TNF-α and IL-6 are key cytokines involved in the amplification of mucosal inflammation, including the activation of macrophages, neutrophils, and T cells, in IBD patients.32 IL-1β is produced by activated macrophages partially through ROS generation34 and plays a key role in the onset of IBD.35 IFN-γ is involved in the perpetuation of intestinal inflammation, which increases the severity of IBD in the colon.36 MCP-1 is involved in the infiltration of immune cells from the bloodstream into the mucosa and submucosa during chronic IBD.37 Moreover, the involvement of IL-17A in the pathogenesis of IBD has recently been explored. IL-17A was known to be produced mainly in a subset of T lymphocytes named Th17. Jiang et al.38 reported that the increase of Th17 cells and Th17-related cytokines was correlated with the elevation of disease activity in IBD patients. 1536

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Arg-Trp and Ile-Gln-Trp, derived from OVT, were found to attenuate TNF-α-induced inflammatory responses and oxidative stress in vascular endothelial cells.44 The underlying molecular mechanisms of anti-inflammatory effect by OVT were not fully investigated, but these findings suggested the possibility of involvement in the prevention of IBD by OVTderived peptides. Further experiments to examine the antiinflammatory effects and underlying mechanisms of OVT and OVT hydrolysates are in progress. In conclusion, we demonstrated for the first time the potent immune-modulating property of the egg white protein ovotransferrin. The administration of OVT prevented the progression of DSS-induced colitis in mice and had a preventative effect by inhibiting the epithelial dysfunction and modulating the cytokine profile. We suggest that OVT may be validated as a potential effective agent in the management of chronic intestinal inflammatory diseases.

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AUTHOR INFORMATION

Corresponding Author

*(Y.M.) Phone: (519) 824-4120. Fax: (519) 824-6631. E-mail: [email protected]. Funding

This study was supported in part by the Advanced Foods and Materials Network (AFMNet), part of the Networks of Centre of Excellence (NCE), Canada, to Y.M., by a grand-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 22248014) to T.M., and by the JSPS Research Fellowship for Young Scientists to Y.K. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Annette Morrison, Maka Natsvlishvili, and Jackie Rombeek for their assistance with the animal studies.

Figure 4. Effect of OVT on colon histology. (A−D) Representative H&E-stained colon sections are shown at 4× magnification. Black scale bar indicates 100 μm for all panels. (E) Colon sections were scored for DSS-induced colonic inflammation and tissue injury as previously described.11 Data represent the mean ± SEM of n = 3. Values without a common letter are significantly different at P < 0.05.

ABBREVIATIONS USED OVT, ovotransferrin; IBD, inflammatory bowel disease; DSS, dextran sodium sulfate; CD, Crohn’s disease; UC, ulcerative colitis; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; TNF, tumor necrosis factor; IL, interleukin; Av-HRP, avidin−horseradish peroxidase; BSA, bovine serum albumin; TMB, 3,3′,5,5′-tetramethylbenzidine; IFN, interferon; MCP, monocyte chemoattractant protein; H&E, hematoxylin and eosin; TGF, transforming growth factor

Ito et al.39 demonstrated that deficient IL-17A gene expression contributed to the prevention of the progression of colitis in DSS-treated mice through the inhibition of MCP-1 expression and neutrophil infiltration. IL-1β,40 IL-6, and transforming growth factor-β (TGF-β)41 were shown to be involved in the induction of Th17 differentiation, which in turn accelerated the secretion of IL-17A associated with neutrophil infiltration. Here, we have demonstrated that OVT treatment significantly reduced the expression of the cytokine proteins (TNF-α and IL-6) and mRNA (TNF-α, IL-6, IL-1β, IFN-γ, MCP-1, and IL17A) in the colonic tissue. The significant decrease of IL-17A protein was not observed in low-dose OVT, but this low dose significantly inhibited the mRNA expression of IL-17A, IL-1β, IL-6, and MCP-1 that might be involved in Th17-related inflammatory pathways and improved the colitic histological score. These results are consistent with other recent studies which demonstrated that dipeptide Ala-Gln42 or the immune milk43 treatment could reduce the Th17-related inflammation responses and improve the histological damage in the colon of DSS-treated mice, suggesting OVT may be involved in the regulation of IL-17A-related inflammatory responses. The OVT hydrolysates produced by acidic or enzymatic digestion have been indicated to possesses higher antioxidant activity than the native protein.19 In addition, tripeptides Ile-

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REFERENCES

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DOI: 10.1021/jf505248n J. Agric. Food Chem. 2015, 63, 1532−1539