The dietary flavonoid myricetin regulates iron

The dietary flavonoid myricetin regulates iron homeostasis by suppressing hepcidin expression Mingdao Mu, Peng An, Qian Wu, Xiaoyun Shen, Dandan Shao, Hao Wang, Yingqi Zhang, Shenshen Zhang, Hui Yao, Junxia Min, Fudi Wang PII: DOI: Reference:

S0955-2863(15)00329-0 doi: 10.1016/j.jnutbio.2015.10.015 JNB 7490

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

22 January 2015 5 October 2015 16 October 2015

Please cite this article as: Mu Mingdao, An Peng, Wu Qian, Shen Xiaoyun, Shao Dandan, Wang Hao, Zhang Yingqi, Zhang Shenshen, Yao Hui, Min Junxia, Wang Fudi, The dietary flavonoid myricetin regulates iron homeostasis by suppressing hepcidin expression, The Journal of Nutritional Biochemistry (2015), doi: 10.1016/j.jnutbio.2015.10.015

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The dietary flavonoid myricetin regulates iron homeostasis by suppressing

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hepcidin expression

Mingdao Mu1, 2, Peng An2, 3, Qian Wu2, Xiaoyun Shen2, Dandan Shao2, Hao Wang2,

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Yingqi Zhang2, Shenshen Zhang1, Hui Yao4, Junxia Min3, Fudi Wang 1,2*

Department of Nutrition, Nutrition Discovery Innovation Institute, College of Public

Health, Zhengzhou University, Zhengzhou 450001, China Department of Nutrition, Nutrition Discovery Innovation Center, Institute of

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Nutrition and Food Safety, School of Public Health, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,

The first affiliated Hospital, Institute for Translational Medicine, School of Medicine,

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Zhejiang University, Hangzhou, Zhejiang 310058, China

Zhejiang University, Hangzhou 310058, China 4

Traditional Chinese Medicine Department, Zhejiang Hospital, Hangzhou 310013,

China

Keywords: Iron-deficiency Anemia; Anemia of Inflammation; Iron Metabolism; Hepcidin; Myricetin

Running Title：Myricetin regulates iron homeostasis


Contact Information: *Address correspondence to: College of Public Health, Zhengzhou University, 100 Science Road, Hangzhou 450001, China; or Department

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of Nutrition, School of Public Health, Zhejiang University, 866 Yuhangtang Road,

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Hangzhou 310058, China. Email: [email protected]; [email protected]

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Abbreviations: AI, anemia of inflammation; BMP, bone morphogenetic protein; ERK, extracellular signal-regulated kinase; ESAs, erythropoiesis-stimulating agents; HAMP, hepcidin antimicrobial peptide; HJV, hemojuvelin; IL-6, interleukin-6; JAK, Janus kinase; LPS, lipopolysaccharide; RES, reticuloendothelial system; ROS, reactive

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oxygen species; SMAD, mothers against decapentaplegic homolog proteins; STAT,

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signal transducer and activator of transcription.

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Financial Support: This work was supported by research grants from Chinese

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National Natural Science Foundation grants (numbers 31530034, 31225013, and 31330036 to F.W. and 31570791 to J.M.) and Zhejiang Provincial Natural Science Foundation (LZ15H160002 to J.M., and Y14H270055 to H.Y.).

Conflicts of interest: None.

Acknowledgments: We are grateful to Dr. Pauline Lee and Dr. Jaroslav Truksa (Scripps Research Institute, La Jolla, CA) for generously providing the HAMP-promoter plasmid. We are also grateful to the members of the Wang laboratory for their encouragement and helpful comments.

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Abstract Hepcidin, a master regulator of iron homeostasis, is a promising target in treatment of

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iron disorders such as hemochromatosis, anemia of inflammation, and iron-deficiency anemia. We previously reported that black soybean seed coat extract could inhibit

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hepcidin expression. Based on this finding, we performed a screen in cultured cells in

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order to identify the compounds in black soybeans that inhibit hepcidin expression. We found that the dietary flavonoid myricetin significantly inhibited the expression of hepcidin both in vitro and in vivo. Treating cultured cells with myricetin decreased both HAMP mRNA levels and promoter activity by reducing SMAD1/5/8

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phosphorylation. This effect was observed even in the presence of bone morphogenic

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protein-6 (BMP6) and interleukin-6 (IL-6), two factors that stimulate hepcidin

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expression. Furthermore, mice that were treated with myricetin (either orally or systemically) had reduced hepatic hepcidin expression, decreased splenic iron levels,

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and increased serum iron levels. Notably, myricetin-treated mice increased red blood cell counts and hemoglobin levels. In addition, pretreating mice with myricetin prevented LPS-induced hypoferremia. We conclude that myricetin potently inhibits hepcidin expression both in vitro and in vivo, and this effect is mediated by altering BMP/SMAD signaling. These experiments highlight the feasibility of identifying and characterizing bioactive phytochemicals to suppress hepcidin expression. These results also suggest that myricetin may represent a novel therapy for treating iron deficiency‒related diseases.

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Introduction Iron is an essential trace element that plays a key role in cellular metabolism. Iron

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deficiency causes anemia and delayed growth and development, whereas excess iron produces reactive oxygen species (ROS), which can damage tissues[1]. Thus,

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systemic iron homeostasis is tightly regulated in order to maintain optimum levels of

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systemic iron[2]. Hepcidin, a circulating hormone produced in and secreted by the liver, was recently identified as a principal regulator of iron homeostasis[3]. Iron absorption, iron recycling, and stored iron mobilization are all coordinated by hepcidin through the degradation of hepcidin’s target, the cellular iron exporter

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ferroportin[4].

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The expression of hepcidin (encoded by the HAMP gene) is regulated by iron stores, inflammation, hypoxia, and erythropoiesis, processes that are regulated primarily by

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the BMP/SMAD and JAK/STAT signaling pathways[5, 6]. Many iron-related disorders are associated with altered hepcidin expression[7]. For example, hepcidin deficiency can cause systemic iron-overload diseases such as hereditary hemochromatosis and iron-loading anemia; on the other hand, increased hepcidin levels have been linked to the pathogenesis of iron-restricted anemia, a common finding in several conditions, including anemia of inflammation, cancer, and chronic kidney disease[8, 9]. Therefore, the hepcidin-ferroportin axis and its related pathways have received increasing interest with respect to the diagnosis and treatment of iron

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disorders[10, 11].

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A growing body of evidence suggests that anti-hepcidin therapies might be beneficial in treating anemia of inflammation, particularly in patients who present with infection

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and/or inflammatory disorders such as autoimmune disease, chronic kidney disease,

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and certain types of cancers[12], all of which are often accompanied by elevated levels of hepcidin. Moreover, several hepcidin antagonists-including dorsomorphin and its derivatives[13, 14], IL-6 antagonists, and the anti-hepcidin spiegelmer oligonucleotide NOX-H94 are reported to have curative effects in models of

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inflammation-associated anemia[15]. However, detailed studies are needed in order to

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determine the therapeutic efficacy and feasibility of hepcidin inhibitors.

For thousands of years, humans have used naturally occurring products for

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healthcare[16]. In recent years, an increasing number of natural product-based drugs have been approved for use in patients[17], inspiring researchers to develop novel, natural approaches for treating iron-related disorders that are caused by excess hepcidin. We previously reported that black soybean seed coat extract inhibits hepcidin expression both in vitro and in vivo[18, 19]. Motivated by this finding, we screened the major bioactive phytochemicals present in black soybean seed coat, including anthocyanins, flavonoids, and polyphenols[20-22]. Our screen revealed that myricetin is a potent inhibitor of hepcidin expression, and given its high clinical

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potential, we used both in vitro and in vivo assays to examine the mechanisms through

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which myricetin modulated hepcidin expression.

Myricetin is a natural, partly cell-permeable dietary flavonoid[23, 24] with a wide

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variety of biological effects, including anti-cancer[25], anti-inflammatory[26], and

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anti-diabetes properties[27]. These beneficial properties are believed to arise from the inhibition of MEK1, the suppression of neoplastic transformation, and the reduction of tumor necrosis factor-alpha (TNF-α, a cytokine that promotes the inflammatory response in inflammatory diseases)[28]. Moreover, cell-free models indicated that

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myricetin possessed iron chelating properties[29, 30]. However, insufficient data are

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available to support the in vivo capacity of myricetin to chelate iron.

Materials and Methods

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Reagents

Myricetin (No. M6760), cyanidin chloride (No. 79457), β-sitosterol (No. S9889), kuromanin chloride (No. 52976), (+)-catechin hydrate (No. C1251), and dimethyl sulfoxide (No. D2650) were all purchased from Sigma Chemical Company. Recombinant human BMP6 (No. 507-BP-020) and recombinant human IL-6 (No. 206-IL-010) were purchased from R&D Systems.

Cell culture

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Human hepatocellular carcinoma cells (HepG2 cells) and human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection. The cells

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were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1X penicillin-streptomycin

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(Gibco); the cells were incubated at 37°C in a humidified atmosphere containing 5%

Measurement of cell viability

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CO2.

Cell viability was evaluated by MTT assay. Briefly, HepG2 cells were cultured in

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DMEM medium as mentioned above in 96-well plates at a density of 1 × 104 cells per

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well. After culturing for 24 h, the cells were treated with various concentrations of

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myricetin for 24 h. Then the medium containing myricetin was removed, and cells were incubated in 100 µL fresh medium supplemented with 10 µL MTT solution

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(5 mg/mL in phosphate buffered solution) for 4 h at 37 °C. After gentle removal of the medium, 100 µL DMSO was added into each well to dissolve the formazan crystals. Absorbance at 570 nm was measured with a microplate reader. The cell viability were estimated as the percentage of the control group.

Luciferase reporter assay The luciferase reporter assay was performed in accordance with the manufacturer’s instructions (Fugene HD Transfection Reagent; Promega). In brief, HEK293 cells

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were plated in 24-well plates at least one day prior to transfection, and the medium was replaced when the cells reached approximately 60% confluence. The

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HAMP-promoter luciferase reporter gene construct pGL3-HAMP was generated, containing a 2.7-kb 5’-flanking genomic region of the human HAMP gene plus the

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5’-UTR (from −2700 to +71 bp), and the cells were co-transfected with pGL3-HAMP

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and the control Renilla reporter construct[31]. Twenty-four hours after transfection, the cells were subjected to a variety of treatments and then lysed in 150 µl luciferase cell culture lysis reagent (Promega). Luciferase activity was measured in the cell lysates using a dual-luciferase reporter assay system (Promega). Relative luciferase

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activity was calculated as the ratio of firefly luciferase to Renilla luciferase[32]. All

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experiments were performed three times.

Quantitative RT-PCR

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RNA was extracted as described previously[33] using the SuperfecTRI Total RNA Isolation Regent (Pufei, Shanghai, China). After synthesizing cDNA using reverse transcriptase, the following primer sequences (5’-3’) were used for qPCR: Human HAMP: forward CAGCTGGATGCCCATGTTC / reverse CAGCAGCCGCAGCAGAA; human ACTIN: forward CACGGCATCGTCACCAACT / reverse CACGCAGCTCATTGTAGAAGGT; mouse Hamp1: forward GCACCACCTATCTCCATCAACA / reverse TTCTTCCCCGTGCAAAGG; mouse Bmp6: forward

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ATGGCAGGACTGGATCATTGC / reverse CCATCACAGTAGTTGGCAGCG; mouse Actb (β-Actin): forward AAATCGTGCGTGACATCAAAGA / reverse

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GCCATCTCCTGCTCGAAGTC.

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Western blot analysis

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The cells were lysed and analyzed as described previously[33], and the phosphorylation levels of SMAD1/5/8, ERK1/2, and STAT3 were measured as described previously[34]. The following primary antibodies were used: rabbit anti-pSMAD1/5/8 (1:1000; Cell Signaling Technology, No. 9511s), rabbit

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anti-SMAD1 (1:1000; Cell Signaling Technology, No. 9743s), rabbit anti-pSTAT3

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(1:1000; Cell Signaling Technology, No. 9131s), rabbit anti-STAT3 (1:1000; Cell

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Signaling Technology, No. 9132s), rabbit anti-pERK1/2 (1:1000; Cell Signaling Technology, No. 4376), rabbit anti-ERK1/2 (1:1000; Cell Signaling Technology, No.

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4695), and mouse anti-β-Actin (1:2000; Sigma-Aldrich, No. A5316).

Animal experiments Adult (6-10-week-old) male C57BL/6 mice (SLRC Laboratory Animal Co., Ltd., Shanghai, China) were housed under pathogen-free conditions with free access to AIN-76A standard mouse diet[35]. The mice in the experimental groups were injected intraperitoneally (i.p.) with either vehicle or myricetin (40 mg/kg body weight) once daily for 1 or 5 days, after which the mice were euthanized under anesthesia (5%

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chloral hydrate, 10 ml/kg body weight by i.p. injection). Whole blood was collected, and the liver and spleen were removed for further analysis. The protocols used to

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measure serum and tissue iron levels have been described previously[33]; in separate experiments, mice received a daily i.p. injection of vehicle or 10 mg/kg myricetin for

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5 days, after which the mice were injected with LPS (5 mg/kg, i.p.); 6 hours after LPS

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injection, the mice were euthanized and analyzed as described above. In another experiment, C57BL/6 mice were fed a diet containing 0.2% (w/w) myricetin for up to 30 days, then sacrificed and analyzed. Each experimental group contained 6-8 mice. All animal experiments were approved by the Institutional Animal Care and Use

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Statistical analysis

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Committee of Zhejiang University.

Group differences were analyzed using ANOVA, and differences between two

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specific groups were examined using Tukey’s post hoc test. Some data were log-transformed to meet the assumption of homogeneity of variances (Bartlett’s test). Where applicable, groups are labeled with letters to indicate significant differences (different letters denote a significant difference). Groups were considered to be significantly different if P