Adenine Inhibits TNF-α Signaling in Intestinal ... - ACS Publications

11 May 2016 - including steroids and mesalazine6 are used for the management of IBD. Considering the ... GE Healthcare Biosciences (Piscataway, NJ, US...
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Adenine Inhibits TNF‑α Signaling in Intestinal Epithelial Cells and Reduces Mucosal Inflammation in a Dextran Sodium Sulfate-Induced Colitis Mouse Model Toshihiko Fukuda,†,§ Kaustav Majumder,† Hua Zhang,† 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, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan S Supporting Information *

ABSTRACT: Adenine (6-amino-6H-purine), found in molokheiya (Corchorus olitorius L.), has exerted vasorelaxation effects in the thoracic aorta. However, the mode of action of the anti-inflammatory effect of adenine is unclear. Thus, we investigated to clarify the effect of adenine on chronic inflammation of the gastrointestinal tract. In intestinal epithelial cells, adenine significantly inhibited tumor necrosis factor-α-induced interleukin-8 secretion. The inhibition of adenine was abolished under the treatment of inhibitors of adenyl cyclase (AC) and protein kinase A (PKA), indicating the effect of adenine was mediated through the AC/PKA pathway. Adenine (5, 10, and 50 mg/kg BW/day) was administered orally for 14 days to female BALB/c mice, and then 5% dextran sodium sulfate (DSS) was given to induce colitis. Adenine (5 mg/kg BW/day) significantly prevented DSS-induced colon shortening, expression of pro-inflammatory cytokines, and histological damage in the colon. These results suggest that adenine can be a promising nutraceutical for the prevention of intestinal inflammation. KEYWORDS: adenine, dextran sodium sulfate, intestinal epithelial cell, pro-inflammatory cytokines, colitis, AC/PKA pathway

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brain, and intestines.29,30 However, there was no evidence of the anti-inflammatory potential of adenine in the colon, although the expression of intestinal AdeR was identified.29 Therefore, in this study, we primarily investigated if adenine possesses an anti-inflammatory potential using both in vitro and in vivo inflammation models.

INTRODUCTION Inflammatory bowel disease (IBD) including ulcerative colitis and Crohn’s disease is a serious intestinal disease.1,2 IBD involves acute and/or chronic inflammatory reactions in intestinal mucosa and the subsequent onset of abdominal pain, diarrhea, and bloody stool. It was reported that IBD was caused by genetic, infective, and environmental factors among others.3−5 However, the etiology and cure for IBD are still unclear. Some medicines including steroids and mesalazine6 are used for the management of IBD. Considering the risk of side effects,7,8 however, it is expected that the application of functional food compounds for the treatment and/or recovery of IBD would be beneficial due to their low risk of side effects. Thus far, it has been reported that some bioactive food compounds including alkaloids,9,10 polyphenols,11,12 proteins,13,14 peptides,15−19 and amino acids20−22 could exhibit anti-colitis activities. It has been reported that Trp-His, an absorbable vasorelaxant dipeptide,23,24 reduced onsets of atherosclerosis25 and colitis.18 These reports allowed us to consider that vasorelaxant natural compounds might also exhibit anti-inflammatory effects. In our previous study, we have demonstrated that adenine (6-amino-6H-purine) found in molokheiya (Corchorus olitorius L.) relaxed contracted rat thoracic aorta in an adenosine receptorindependent manner.26 Furthermore, recent studies demonstrated that adenine could play a role in neuroprotection27 and nociception,28,29 suggesting that adenine can act not only as a purine base but also as a signaling molecule. The adenine receptor (AdeR), which was recently discovered and characterized as a member of the family of G protein-coupled purinergic receptors, is expressed in diverse organs including the lungs, kidney, liver, © XXXX American Chemical Society

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MATERIALS AND METHODS

Materials. Adenine was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dextran sodium sulfate (DSS, MW 36−50 kDa) was purchased from MP Biomedicals (Solon, OH, USA). Tumor necrosis factor (TNF)-α was obtained from Life Technologies (Carlsbad, CA, USA). H-89 and SQ22536 were obtained from Abcam Inc. (Cambridge, MA, USA). Horseradish peroxidase (HRP) conjugated streptavidin and ECL prime detection reagents were obtained from GE Healthcare Biosciences (Piscataway, NJ, USA). All other chemicals were of analytical reagent grade and were used without further purification. Cell Culture. Human intestinal epithelial cells (Caco-2 cells; ATCC, Manassas, VA, USA) were cultured with 20% fetal bovine serum (FBS; HyClone, Logan, UT, USA)/DMEM-F12 (Life Technologies) supplied with 1 mM sodium pyruvate (Life Technologies) and 50 U/mL penicillin−streptomycin (Life Technologies). Caco-2 cells were maintained at 37 °C in a humidified 5% CO2 incubator, and cells at passage 15−40 were used in this study. Measurement of TNF-α-Induced Interleukin-8 Production in Caco-2 Cells. To evaluate the anti-inflammatory effect of adenine Received: February 9, 2016 Revised: April 13, 2016 Accepted: May 4, 2016

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DOI: 10.1021/acs.jafc.6b00665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry in vitro, interleukin (IL)-8 secretion in Caco-2 cells was measured. Caco-2 cells were first seeded on a 48-well plate (Corning, Corning, NY, USA). After ∼80% confluence, Caco-2 cells were washed twice with phosphate-buffered saline (PBS). Caco-2 cells were treated with adenine (0.1, 0.5, 1, 3, 5, or 10 mM) for 2 h and then stimulated by TNF-α (2 ng/mL) for 4 h at 37 °C. After treatment, cell supernatant was collected and ELISA experiment was performed for measurement of IL-8 concentration. ELISA plates (Corning) were coated with antihuman IL-8 (50 ng/well in 100 μL of 100 mM sodium phosphate buffer, pH 9.0 (BD Bioscience San Jose, CA, USA)) overnight at 4 °C. After the plate had been washed with PBS−0.05% Tween-20 (PBS-T), the plate was blocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at 37 °C. The supernatant and standards were added to the plate and incubated for 2 h at 37 °C. After washing, biotin IL-8 antibody (1:500 v/v; BD Bioscience) was incubated for 1 h at 37 °C. After washing, 100 μL of avidin−HRP conjugate (1:2000 v/v; BD Bioscience) was added for 30 min at 37 °C. After washing, color development was performed by adding 50 μL/well of 3,3′,5,5′tetramethylbenzidine (TMB; Sigma-Aldrich) and stopped with 25 μL/well of 0.25 M H2SO4. The absorbance was read at 450 nm using a plate reader (iMark model 550, Bio-Rad, Richmond, CA, USA). Cell Viability Assay. Cell viability was evaluated by WST-1 Cell Proliferation Reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s protocol. Viability was shown as a percent relative to control cells. Western Blot Analysis. Caco-2 cells were seeded on a 6-well plate (Corning). After ∼80% confluence, Caco-2 cells were washed twice with PBS. Caco-2 cells were treated with adenine (1 mM) for 2 h and then stimulated by TNF-α (2 ng/mL) for 1 h at 37 °C. Treated cells were washed with cold PBS twice and lysed in radioimmunoprecipitation assay (RIPA) buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 14000g for 15 min at 4 °C. The protein concentration was measured by DC protein assay (Bio-Rad). The protein extract was mixed with an equal volume of sample buffer (20% glycerol, 4% SDS, 3% dithiothreitol, 0.002% bromophenol blue, and 0.125 M Tris-HCl, pH 6.8) and maintained at 100 °C for 10 min. An aliquot of sample in buffer (20 μg protein/lane) was applied to a 10% SDS-PAGE gel for 1.5 h at 20 mA and transferred onto a nitrocellulose membrane (Bio-Rad) for 1.5 h at 40 mA. The membrane was blocked using 5% BSA in Tris-buffered saline (TBS) and incubated with primary antibody (anti-phospho-c-Jun N-terminal kinase (p-JNK), anti-JNK, antiphospho-IκBα (p-IκBα) (Cell Signaling Technology, Inc., Danvers, MA, USA)) and anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), at a dilution of 1:1000 v/v, overnight at 4 °C, and then incubated with HRP-conjugated secondary antibody (Promega, Madison, WI, USA), at a dilution of 1:10000 v/v, for 1 h at room temperature. The blots on membrane were detected using ECL Western Blotting Detection Reagent. RNA Isolation and Real-Time RT-PCR. Total RNA in cells was extracted using an Aurum Total RNA Mini Kit (Bio-Rad) according to the manufacturer’s protocol. The quantity and quality of the extracted RNA were determined using Nano Drop ND-1000 (Thermo Scientific). The cDNA was synthesized by the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific), according to the manufacturer’s protocol. Real-time quantitative PCR was performed using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) on a MyiQ single-color real-time PCR detection system (Bio-Rad) under the following conditions: denaturation for 15 s at 95 °C, annealing for 30 s at 60 °C, and extension for 30 s at 72 °C (40 cycles). Primers (Supporting Information Table S1) were synthesized by the University of Guelph, Laboratory Services Molecular Biology Section (Guelph, ON, Canada). Relative gene expression was calculated using the 2−ΔΔCt method31 and β-actin as the reference gene. Induction and Treatment of Colitis in Mice. Female 6−8-weekold BALB/c mice (Charles River Laboratories, Montreal, QC, Canada) were randomly divided into groups (n = 8/group), housed on a 12 h light/dark cycle, and allowed to access to standard food and water ad libitum. Adenine was added to drinking water at

concentrations of 0.05, 0.1, and 0.5 mg/mL, equivalent to doses of 5, 10, and 50 mg/kg body weight (BW) for 22 days, respectively. On day 15, DSS (5%, w/v) was added to drinking water to induce mucosal inflammation and continued for the following 8 days. The negative control group received water only, and the positive control group received water with DSS for 8 days. A sample vehicle group received adenine (50 mg/kg BW) only, but no DSS. After the experiment, mice were euthanized and colons were collected and sectioned longitudinally. Colon samples for ELISA were flash frozen with liquid nitrogen and stored at −80 °C until use. Colon samples for RT-PCR were put in RNAlater (Life Technologies) and stored at −80 °C until use. 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 (AUP no. 1536). Cytokine ELISAs in Colon Tissue. The extracted colons were homogenized with 5 times volume 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) on ice for 3 min using a homogenizer. The supernatants were collected following centrifugation at 14000g for 15 min at 4 °C. The protein concentration was measured by DC protein assay. TNF-α concentration was measured using a mouse TNF-α ELISA Ready-SET-Go! kit (eBioscience, San Diego, CA, USA), according to the manufacturer’s protocol. IL-6 concentration was measured as described previously.14 Briefly, ELISA plates (Corning) were coated with anti-mouse IL-6 (50ng/well; BD Bioscience, catalog no. 554400) overnight at 4 °C. After the plate had been washed with PBS-T, it was blocked with 1% BSA-containing PBS for 1 h at 37 °C. The sample protein and standards were incubated for 2 h at 37 °C. After washing, biotin IL-6 antibody (1:500 v/v; BD Bioscience) was incubated for 1 h at 37 °C. After washing, avidin− HRP conjugate (1:2000, v/v) was added for 30 min at 37 °C, and color development was performed by adding 50 μL/well of TMB and stopped with 25 μL/well of 0.25 M H2SO4. The absorbance was read at 450 nm by a plate reader (iMark model 550). Histological Analysis. Histological analysis was performed as described previously.14 A section of the distal colon was fixed in buffered formalin (Fisher Scientific). Paraffin-embedded sections were stained with hematoxylin and eosin (H&E; Animal Health Laboratory, University of Guelph, Guelph, ON, Canada). Photographs of the stained samples were taken using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). The stained samples were assessed by a pathologist double-blinded to treatment for injury and inflammation using a scoring system as used in the previous study14 and indicated in Table S2 of the Supporting Information. Sections were scored for the degree, extent, and depth of inflammation, as well as the amount of damage to the glands and epithelium. Grades were added to give a total histologic score ranging from 0 to 19. Statistical Analysis. The results were expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad, San Diego, CA, USA). Statistical differences were determined by one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison or Bonferroni’s for post hoc analysis, respectively. P < 0.05 was considered statistically significant.

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RESULTS Adenine Inhibited TNF-α Induced Expression of Proinflammatory Cytokines in Caco-2 Cells. To examine the anti-inflammatory effect of adenine in vitro, Caco-2 cells were treated with adenine, followed by TNF-α (2 ng/mL) treatment to induce inflammation. As shown in Figure 1, adenine inhibited TNF-α-induced IL-8 secretion in a dose-dependent manner without cytotoxicity in the experimental condition. To further test the effect of adenine, mRNA expression levels of proinflammatory cytokines were evaluated by RT-PCR. As shown in Figure 2, gene expression levels of IL-1β, IL-8, and TNF-α B

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were significantly elevated by TNF-α stimulation compared with the control and clearly reduced by treatment with adenine (1 mM). These results indicate that adenine could exhibit an anti-inflammatory effect in intestinal tissue. Adenine Inhibited TNF-α-Induced IL-8 Secretion through Adenyl Cyclase/Protein Kinase A Pathway. To further examine the involvement of the AdeR pathway on the adenine-induced anti-inflammatory effect, Caco-2 cells treated with adenyl cyclase (AC) and protein kinase A (PKA) inhibitors were evaluated. Caco-2 cells were pretreated with SQ22536 (100 μM, AC inhibitor) or H-89 (20 μM, PKA inhibitor) for 30 min prior to adenine (1 mM) treatment, respectively. Then, TNF-α (2 ng/mL) was added and incubated to induce inflammation. The inhibition of IL-8 by adenine was clearly attenuated in the presence of SQ22536 (Figure 3A). A similar result was observed in the presence of H-89 (Figure 3B). These results indicate that adenine inhibited TNF-α-induced IL-8 secretion through the AC/PKA pathway. TNF-α stimulation activates mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways32 and subsequently induces IL-8 secretion.33 Thus, to assess the involvement between the adenine-induced anti-inflammatory effect and TNF-α-induced signaling cascade, p-JNK and p-IκBα were evaluated by Western blot. As shown in Figure 4, Caco-2 cells stimulated by TNF-α had significantly elevated p-JNK and p-IκBα, and the treatment of adenine significantly inhibited both phosphorylations. These results indicate that the adenine-inducing anti-inflammatory

Figure 1. Effect of adenine on TNF-α-induced IL-8 secretion in Caco-2. (A) Adenine was pretreated in Caco-2 cells for 2 h, and then TNF-α was added and incubated for 4 h. IL-8 concentration was measured by ELISA. (B) Cell viability was evaluated by WST-1. Results are expressed as the mean ± SEM (n = 4−5).

Figure 2. Effect of adenine on TNF-α-induced cytokine gene expression in Caco-2. mRNA expression of pro-inflammatory cytokines (A) TNF-α, (B) IL-1β, (C) IL-8, and (D) IL-10 was measured by RT-PCR. Adenine (1 mM) was pretreated in Caco-2 cells for 2 h, and then TNF-α was added and incubated for 4 h. Results are expressed as the mean ± SEM and as a relative expression to negative control (n = 3). Values without a common letter are significantly different at P < 0.05. C

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and ulceration, neutrophil infiltration, colon shortening, and diarrhea.34 Thus, the DSS-induced colitis mouse model was used in the present study to confirm the anti-inflammatory activity of adenine in vivo. As shown in Figure 5B, the ratio of body weight

Figure 3. Involvement of AC and PKA on adenine-induced antiinflammatory effect in Caco-2. (A) AC inhibitor (SQ22536, 100 μM) was pretreated prior to adenine (1 mM) treatment. After 2 h of incubation, TNF-α was added and incubated for 30 min. (B) PKA inhibitor (H-89, 20 μM) was pretreated prior to adenine (1 mM) treatment. After 2 h of incubation, TNF-α was added and incubated for 4 h. TNF-α-induced IL-8 secretion was measured by ELISA. Results are expressed as the mean ± SEM (n = 3−6).

Figure 5. Effect of adenine on weight loss in DSS-induced colitis mice. (A) Mice were given 5, 10, and 50 mg/kg BW adenine for 22 days. On day 15, colitis was induced with 5% DSS added to drinking water. (B) Mice were weighed daily, and changes in body weight are reported as percent change relative to the first day of DSS treatment. Data represent means ± SEM (n = 8). Unless indicated, no significant difference was observed between groups. Values without a common letter are significantly different at P < 0.05.

effect should involve the suppression of the TNF-α-mediated MAPKs- and NF-κB-related signalings through activation of the AC/PKA pathway in Caco-2 cells. Effect of Adenine on DSS-Induced Colitis in Mice. Administration of DSS produces similar symptoms observed in ulcerative colitis in humans, including mucosal inflammation

change after DSS treatment was significantly decreased after 5 days of DSS treatment in the DSS group compared with the negative control group. All adenine treatment groups also had

Figure 4. Effect of adenine on phosphorylations of JNK and IκBα in Caco-2. Adenine (1 mM) was pretreated in Caco-2 cells for 2 h, and then TNF-α was added and incubated for 1 h. The expressions of phosphorylated JNK and IκBα in Caco-2 cells were determined by Western blotting. The respective means of p-JNK/JNK and p-IκBα/IκBα are shown as a ratio relative to the negative control, respectively. Data represent the mean ± SEM (n = 4). Values without a common letter are significantly different at P < 0.05. D

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Journal of Agricultural and Food Chemistry significantly decreased body weight compared with the negative control group. The adenine-50 group significantly lost body weight compared with the DSS group after 5, 7, and 8 days of DSS treatment. However, the adenine vehicle group did not show any weight decrement. After the experiment, the DSSinduced colon shortening was confirmed as an indicator of the severity of DSS-induced colitis in mice.34 As shown in Figure 6,

Figure 7. Effect of adenine on the expression of pro-inflammatory cytokines in DSS-treated mouse colon tissue. Protein expression of (A) TNF-α and (B) IL-6 was measured by ELISA. Results were expressed as picograms of cytokine relative to protein concentration in the colon. Data represent the mean ± SEM (n = 8). (∗) P < 0.05 and (∗∗) P < 0.01 versus DSS group.

Figure 6. Effect of adenine on colon length in DSS-induced colitis mice. After the animal experiment, excised colon length was measured. Data represent means ± SEM (n = 8). (∗) P < 0.05 and (∗∗) P < 0.01 versus DSS group.

staining and scoring. As shown in Figure 8A, the DSS group showed clearly severe epithelial damage, depletion of the goblet cells, thickening of the mucosa, cellular infiltration into the submucosa and lamina propria, and destruction of the architecture compared with the negative control group. Although the adenine-50 group did not show histological improvement, the adenine-5 group showed a mild loss of epithelial cells and less depletion of the goblet cells. A similar result was confirmed in histological scoring (Figure 8B).

the DSS group had significant colon shortening compared with the negative control group. In contrast, the adenine-5 group showed significant prevention of the shortening, although the adenine-10 and -50 groups did not. The adenine vehicle group did not exhibit colon shortening. To further investigate the anticolitis effect of adenine, TNF-α and IL-6 in colon tissue, which play key roles in the initiation and amplification of inflammatory responses that lead to intestinal injury,35 were evaluated. Both TNF-α and IL-6 were significantly increased in the DSS group compared with the negative control group. All adenine groups had significantly reduced TNF-α and IL-6 protein levels (Figure 7). These results suggest that adenine has the potential to prevent colitis through anti-inflammatory effects, although adenine did not recover clinical symptoms. Note that AdeR mRNA in the colon was detected by RT-PCR; however, expression levels between adenine treatment groups were not significantly different (Supporting Information Figure 1). Effect of Adenine on Histological and Morphological Damage in DSS-Treated Mice. Inflamed colon is histologically confirmed with typical damages such as severe lesions in the mucosa, infiltration of neutrophils, and lymphocyte infiltration among others.36 Colon histological evaluation for negative, DSS, and adenine-5 and -50 groups was performed by H&E

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DISCUSSION In the present study, we have demonstrated that adenine exerts an anti-inflammatory effect in Caco-2 cells, and this mechanism may be through the AC/PKA signaling pathway by AdeR activation. In vivo, using DSS-induced colitis mice, the administration of adenine (5 mg/kg BW) prevented colon shortening and morphological damage. In addition, all adenine treatment groups showed significantly reduced pro-inflammatory cytokine expression in colon tissue. During the onset of IBD, mucosal immune responses are impaired due to various factors such as genetic and environmental factors among others.3−5 Thereafter, the mucosal barrier is destroyed, leading to intestinal inflammation. Subsequently, activated immune cells overproduce pro-inflammatory cytokines. In IBD patients, TNF-α, IL-6, and IL-1β were detected as critical E

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hamster ovary cells.42 Thus, to clarify the adenine-induced signaling pathway(s) in Caco-2, the relationship of the AC/PKA signaling with TNF-α-induced IL-8 secretion was examined. Per the results, IL-8 inhibition by adenine was clearly abolished by the treatment of AC and PKA inhibitors (Figure 3), indicating that adenine exhibited an anti-inflammatory effect through AC/PKA activation. Because the activation of the PKA pathway inhibits JNK44,45 and IκBα,46 the crosstalk between the AC/PKA and TNF-α-activated cascades was supported. As it has been reported that AdeR in hepatic stellate cells was involved with PKA,43 the activation of the AC/PKA signaling pathway by adenine might be through AdeR activation. To clarify the anti-inflammatory potential of adenine in vivo, the DSS-induced colitis mouse model was examined. Treatment with adenine (5 mg/kg BW) clearly reduced colon shortening, pro-inflammatory cytokines, and histological damage in the colon, despite no prevention of weight loss. These results, except weight changes, are in good agreement with previous studies, which demonstrated that a heterocyclic alkaloid,9 amino acid,20 and peptide18 could reduce colitis symptoms. The high dose (50 mg/kg BW) of adenine did not reduce weight loss or histological damages. However, the adenine vehicle group did not show any negative symptoms such as weight loss, colon shortening, or pro-inflammatory cytokine expression in colon, indicating that adenine itself does not induce any colitis symptoms. Although the inhibition of pro-inflammatory cytokines by the high dose (10 and 50 mg/kg BW) of adenine was clearly confirmed, it is difficult to conclude actual intake effects of adenine because the present in vivo study was limited to evaluating detailed mechanism(s). In the immune system, macrophages initially recognize and respond against foreign pathogens.47,48 Subsequently, pro-inflammatory mediators secreted from macrophages develop intestinal inflammation. Therefore, there might be other potential effects of adenine, which need to be further examined. Aherne et al. demonstrated that the adenosine receptor agonist that activates PKA signaling could provide

Figure 8. Effect of adenine on colon histology. (A) Representative H&E-stained colon sections are shown. (B) Colon sections were scored for DSS-induced colonic inflammation and tissue injury as previously described under Materials and Methods. Data represent the mean ± SEM (n = 8). Values without a common letter are significantly different at P < 0.05.

cytokines for mucosal immune responses.37 TNF-α, which is produced as membrane-bound TNF-α and then released by TNF-converting enzyme as a soluble TNF-α, plays a key role in intestinal inflammation. Caco-2 cells produce cytokines such as IL-8 in response to TNF-α stimulation. Here, we found that adenine inhibited TNF-α-induced IL-8 secretion in Caco-2 cells (Figure 1A). In a further investigation focused on gene expression by RT-PCR, adenine inhibited mRNA expression of IL-1β, IL-8, and TNF-α (Figure 2). These results were consistent with previous studies that reported peptides inhibited IL-8 secretion and reduced gene expression of pro-inflammatory cytokines in Caco-2 cells.19,38 In addition, the fact that the anti-inflammatory effect was confirmed in DSS-treated mice suggests that adenine may have a beneficial potential for treatment of IBD. TNF-α activates MAPKs and NF-κB signaling that play a crucial role in intestinal inflammation.39,40 Treatment of adenine reduced JNK and IκBα phosphorylations (Figure 4), which are highly detected in intestinal tissue in IBD.39,41 These results were concordant with reports that small peptides18,38 that inhibited IL-8 secretion could inhibit TNF-α-stimulated p-JNK and p-IκBα in intestinal epithelial cells. Because of the anti-inflammatory activity of adenine in vitro, adenine could act as an anti-inflammatory signaling molecule. Recent studies demonstrated that adenine can play a role in neuroprotection27 and nociception28,29 through AdeR, which was identified as a G protein-coupled purinergic receptor.42 Watanabe et al. reported that adenine induced cell stellation in hepatic stellate cells.43 In addition, the stellation was abrogated with PKA inhibition, suggesting that adenine acts with PKA activation. Meanwhile, Thimm et al. reported that adenine inhibited AC and then reduced cAMP level in AdeR-transfected Chinese

Figure 9. Schematic diagram of the possible anti-inflammatory effect of adenine. F

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Journal of Agricultural and Food Chemistry mucosal protection in the IBD model.49 Furthermore, Lee et al. reported that caffeine, having a purine structure similar to that of adenine, prevented DSS-induced colitis through improvement of the intestinal barrier, by suppressing chitinase 3-like 1.50 Because it has been reported that caffeine also involves the PKA signaling pathway,51 adenine may possess not only an anti-inflammatory effect but also protection of the intestinal barrier and its related signaling. Furthermore, in our previous study, we demonstrated that the imidazole moiety in adenine was important for vasorelaxation.26 By considering these reports, purines containing the imidazole moiety might be key to investigate for bioactive functionalities. Further experiments focusing on structure− activity are expected. In conclusion, we have found that adenine can exhibit an antiinflammatory effect through AC/PKA activation in intestinal epithelial cells (Figure 9). Furthermore, the effect of adenine was clearly confirmed in vivo using DSS-induced colitis mice. This study first provides a point of view that adenine could act as a signaling molecule in intestinal tissues. In addition, this study suggests that adenine may be a potential therapeutic agent for the treatment of intestinal inflammation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00665. Figure S1 and Tables S1 and S2 (PDF)

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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 Japan Society for the Promotion of Science Research Fellowship for Young Scientists to T.F. (No. 14J03039). We also acknowledge funding support from the Advanced Foods and Materials Network (AFMNet). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AdeR, adenine receptor; AC, adenyl cyclase; BSA, bovine serum albumin; BW, body weight; DSS, dextran sodium sulfate; H&E, hematoxylin and eosin; IBD, inflammatory bowel disease; IL, interleukin; JNK, c-jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; NF-κB, nuclear factor-κB; PBS, phosphate-buffered saline; PKA, protein kinase A; PMSF, phenylmethanesulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; TMB, 3,3′,5,5′-tetramethylbenzidine substrate solution; TNF, tumor necrosis factor

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REFERENCES

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