Article pubs.acs.org/JAFC
Inhibitory Effects of 4′-Demethylnobiletin, a Metabolite of Nobiletin, on 12‑O‑Tetradecanoylphorbol-13-acetate (TPA)-Induced Inflammation in Mouse Ears Xian Wu,† Mingyue Song,† Kanyasiri Rakariyatham,† Jinkai Zheng,†,# Minqi Wang,† Fei Xu,† Zili Gao,† and Hang Xiao*,†,§ †
Department of Food Science, University of Massachusetts, Amherst, Massachusetts, United States College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan, People’s Republic of China # Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China §
ABSTRACT: Nobiletin (NOB) is major citrus flavonoid with many health-promoting benefits. We reported previously that 4′demethylnobiletin (4DN), a major metabolite of NOB, significantly inhibited lipopolysaccharide (LPS)-stimulated inflammation in RAW 264.7 macrophages. In this study, we further studied the anti-inflammatory effects of 4DN in TPA-induced skin inflammation in mice. We demonstrated that topical application of 4DN decreased TPA-induced ear edema by >88 ± 4.77% in mice. This inhibitory effect was associated with inhibition on TPA-induced up-regulation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Immunoblotting results showed that 4DN resulted in profound effects on multiple proteins related with inflammation and carcinogenesis. 4DN significantly decreased the expression levels of iNOS, COX-2, and MMP-9, suppressed phosphorylation of PI3K/Akt and ERK, and increased the levels of HO-1 and NQO1 in TPA-treated mice. Overall, the results demonstrated that 4DN had strong anti-inflammatory effects in vivo, which provided a scientific basis for using NOB to inhibit inflammation-driven diseases. KEYWORDS: 4′-demethylnobiletin, nobiletin, anti-inflammatory, 12-O-tetradecanoylphorbol-13-acetate, ear edema
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extract.6 Previous studies have demonstrated a wide range of biologically beneficial activities of NOB, including antiinflammation,7,8 antitumor,9,10 and antiatherosclerosis.11 A growing body of evidence suggests that biotransformation plays critical roles in the bioactivities of dietary compounds.12,13 The metabolites generated in the body by the biotransformation may have more potent bioactivities in comparison with the parent compounds.14,15 After dietary intake, NOB was biotransformed into its demethylated metabolites, such as 4′demethylnobiletin (4DN), 3′-demethylnobiletin, and 3′,4′didemethylnobiletin.8 We reported previously that 4DN was the most abundant metabolite of NOB found in the mouse colonic mucosa after long-term dietary administration of NOB in mice, and its colonic level was much higher than that of NOB.16 Particularly, dietary administration of NOB (0.05 wt %) for 20 weeks resulted in about 24 nmol/g of tissue of 4DN in colonic mucosa. In contrast, the level of NOB was only about 2 nmol/g tissue. The level of 4DN was about 11-fold higher than that of NOB in mouse colon.16 Therefore, it is likely that 4DN may play an important role in eliciting the biological effects of orally administered NOB, such as anti-inflammatory effects in the colon against colitis and colon carcinogenesis. The anti-inflammatory effects of NOB have been extensively studied using several in vivo and in vitro models. In vitro
INTRODUCTION Epidemiological studies have suggested that vegetable and fruit consumption is associated with lowered risk of various diseases, such as cancer and obesity. The health beneficial effects of natural products can be attributed to the bioactive phytochemicals present in these foods.1 Using phytochemicals has various advantages, because very few unwanted side effects have been reported during animal and human studies and they usually target multiple signaling pathways.2 Due to the unique flavors and nutritional values, citrus fruits, such as oranges, tangerines, lemons, limes, and grapefruits, are widely consumed and have become an important part of our diet. Citrus peels are processed to obtain the volatile and nonvolatile fractions in the food, pharmaceutical, and cosmetic industries. In some Asian countries, aged citrus peels have been utilized as traditional medicinal resources since ancient times to treat various diseases, such as abdominal fullness and distention, cough, stomach upset, skin inflammation, and hypertension.3 Fresh citrus fruits are good sources of dietary fibers and carbohydrates. In addition to these macronutrients, citrus fruits contain significant amounts of vitamins and phytochemicals, including flavonoids, carotenoids, and limonoids.4 Due to the antioxidant activities of these constituents, they are of great importance in the health beneficial effects of citrus fruits.5 Polymethoxyflavones (PMFs), a unique class of citrus flavonoids, are abundantly and almost exclusively found in the peel of sweet oranges (Citrus sinensis) and mandarin oranges (Citrus reticulata). Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone, NOB) is a major citrus PMF present in orange peel © 2015 American Chemical Society
Received: Revised: Accepted: Published: 10921
October 25, 2015 December 2, 2015 December 10, 2015 December 10, 2015 DOI: 10.1021/acs.jafc.5b05156 J. Agric. Food Chem. 2015, 63, 10921−10927
Article
Journal of Agricultural and Food Chemistry
Figure 1. (A) Chemical structures of nobiletin (NOB) and 4′-demethylnobiletin (4DN), a major metabolite of nobiletin; HPLC chromatograms of NOB (B) and 4DN (C) indicating purity of >98%.
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studies demonstrated that NOB inhibited LPS-induced overexpression of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophage cells17 and gene expression of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in J774A.1 macrophages.18 In vivo study showed that NOB inhibited TPA-induced skin inflammation in mice via inhibiting NO, O2−, and PGE2 generation and reducing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression.7 Recently, we demonstrated that 4DN, the major metabolite of NOB, effectively inhibited LPS-induced inflammatory response in RAW 264.7 macrophages, which was evidenced by reduced production of NO, PGE2, IL-1β, and IL-6 and suppressed gene expression of iNOS and COX-2, as well as elevated gene expression of antioxidant enzymes heme oxygenase-1 (HO-1) and NADH quinone oxidoreductase 1 (NQO1).19 However, information on the in vivo anti-inflammatory effects of 4ND and its molecular mechanisms remains unavailable. Therefore, the aim of this study was to determine the inhibitory effects of 4DN on TAP-stimulated skin inflammation in mice and elucidate the underlying molecular mechanisms. We selected the skin inflammation mouse model to determine the in vivo effects of 4DN because topical application of 4DN can elucidate the direct effects of 4DN itself rather than those produced by the metabolites of 4DN as may be seen in the model in which oral administration of 4DN is used.
MATERIALS AND METHODS
Chemicals. NOB was purchased from Quality Phytochemicals LLC (Edison, NJ, USA). 4DN was chemically synthesized as previously described.15,20,21 Chemical structures of NOB and 4DN are illustrated in Figure 1A. The purity of NOB and 4DN is >98% according to the HPLC analysis as shown in Figure 1B,C.15,20,21 12-OTetradecanoylphorbol-13-acetate (TPA) was purchased from SigmaAldrich (St. Louis, MO, USA), and acetone was obtained from Fisher Scientific (Hampton, NH, USA). Animals, Diets, and Treatments. The protocol for the animal experiment was approved (permission no. 2011-0066) by the Institutional Animal Care and Use Committee of the University of Massachusetts Amherst. Male CD-1 (5−6 weeks old) mice were purchased from Charles River Breeding Laboratories (Kingston, NY, USA). The mice were kept in our animal facility for at least 1 week for acclimation before use. Upon arrival, mice were kept in a temperaturecontrolled animal room (23 °C) with free access to water and AIN93G diet and kept on a 12 h light/12 h dark cycle. TPA-Induced Skin Inflammation in Mouse. Mice were randomly divided into six groups (six animals each). Group 1, negative control: both ears of CD-1 mice in group 1 were topically treated with 10 μL of acetone (vehicle) acetone 30 min prior to application of another acetone. Group 2, TPA control: both ears of CD-1 mice in group 2 were topically treated with 10 μL of acetone 30 min prior to application of 2 nmol of TPA in 10 μL of acetone. Groups 3−6, treatment groups: both ears of CD-1 mice in treatment groups 3−6 were topically treated with NOB (2 or 4 μmol) or 4DN (2 or 4 μmol) in 10 μL of acetone 30 min prior to application of 2 nmol of TPA in 10 μL of acetone. All mice were sacrificed 6 h after TPA (acetone for group 1) treatment. Ear punches (6 mm in diameter) were taken immediately, weighed, frozen, and stored at −80 °C. 10922
DOI: 10.1021/acs.jafc.5b05156 J. Agric. Food Chem. 2015, 63, 10921−10927
Article
Journal of Agricultural and Food Chemistry ELISA for Cytokines. Ear tissues were homogenized in a phosphate buffer solution containing 0.4 M NaCl, 0.05% Tween-20, 0.5% BSA, 0.1 mM benzethonium, and 1% protease inhibitor cocktail (Boston Bioproducts, Boston, MA, USA). The homogenates were centrifuged at 12000g for 30 min at 0 °C. The supernatant fraction was used for the determination of cytokine, IL-1β, IL-6, and TNF-α protein levels in mouse ears by ELISA kits, according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Immunoblotting Analysis. Ear tissues were homogenized in RIPA lysis buffer containing 1% protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Boston Bioproducts). The homogenates were centrifuged at 12000g for 20 min at 0 °C. Protein concentrations were determined using the BCA method (Pierce, Rockford, IL, USA). The supernatant fraction was subjected to immunoblot analysis as we previously described.17 Antibodies for iNOS, COX-2, PI3K, p-Akt, Akt, p-ERK, ERK, and HO-1 were obtained from Cell Signaling Technology (Beverly, MA, USA). p-PI3K and NQO1 were obtained from Santa Cruz (Santa Cruz, CA, USA). Anti β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA). Statistical Analysis. Data are presented as means ± standard error (SE) or standard deviation (SD) for the indicated number of independently performed experiments. Student’s t test was used to assess the mean difference between two groups. An analysis of variance (ANOVA) model was used for the comparison of differences among three or more groups. A p value of
