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Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes
Accepted Manuscript Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes Jin Kyeong Choi, Yong Hyun Jang, Soyoung Lee, Sang-Rae Lee, Young-Ae Choi, Meiling Jin, Jung Ho Choi, Jee Hun Park, Pil-Hoon Park, Hyukjae Choi, Taeg Kyu Kwon, Dongwoo Khang, Sang-Hyun Kim PII:
S0278-6915(17)30623-3
DOI:
10.1016/j.fct.2017.10.025
Reference:
FCT 9350
To appear in:
Food and Chemical Toxicology
Received Date: 3 July 2017 Revised Date:
10 October 2017
Accepted Date: 13 October 2017
Please cite this article as: Choi, J.K., Jang, Y.H., Lee, S., Lee, S.-R., Choi, Y.-A., Jin, M., Choi, J.H., Park, J.H., Park, P.-H., Choi, H., Kwon, T.K., Khang, D., Kim, S.-H., Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes, Food and Chemical Toxicology (2017), doi: 10.1016/ j.fct.2017.10.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chrysin attenuates atopic dermatitis by suppressing inflammation of
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keratinocytes
Jin Kyeong Choi a, Yong Hyun Jang b, Soyoung Lee c, Sang-Rae Lee d, Young-Ae Choi a, Meiling Jin a, Jung Ho Choi e, Jee Hun Park e, Pil-Hoon Park f, Hyukjae Choi f, Taeg Kyu Kwon g,
Cell & Matrix Research Center, Department of Pharmacology, bDepartment of Dermatology,
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Dongwoo Khang h,*, Sang-Hyun Kim a,*
School of Medicine, Kyungpook National University, Daegu, Republic of Korea c
Immunoregulatory Materials Research Center, Jeongeup, dNational Primate Research Center,
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Republic of Korea R&D Center Pharmaceutical lab, Korean Drug Co., LTD, Seoul, Republic of Korea
f
College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea Department of Immunology, School of Medicine, Keimyung University, Daegu, Republic of
Korea
Department of Physiology, School of Medicine, Gachon University, Incheon, Republic of Korea
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Correspondence to: Prof. Sang-Hyun Kim, Department of Pharmacology, School of Medicine, Kyungpook National University, 101 Dongin-dong, Jung-gu, Daegu 700-422, Republic of Korea. E-mail: [email protected]; Tel: +82-53-420-4838; Fax: +82-53-423-4838 and Prof. Dongwoo Khang, Department of Physiology, School of Medicine, Gachon University, Incheon 406-840, Republic of Korea E-mail: [email protected]; Tel: +82-32-897-6515; Fax: +82-32-8996515. 1
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Abstract
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We previously reported the inhibitory effect of chrysin, a natural flavonoid plentifully contained in propolis, vegetables and fruits, on the mast cell-mediated allergic reaction. In this study, we evaluated the effect of chrysin on atopic dermatitis (AD) and defined underlying mechanisms of
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action. We used an AD model in BALB/c mice by the repeated local exposure of 2,4dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae extract, DFE) to
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the ears. Repeated alternative treatment of DNCB/DFE caused AD-like skin lesions. Oral administration of chrysin diminished AD symptoms such as ear thickness and histopathological analysis, in addition to serum IgE and IgG2a levels. Chrysin decreased infiltration of mast cells, and reduced serum histamine level. Chrysin also suppressed AD by inhibiting the inflammatory responses of Th1, Th2, and Th17 cells in mouse lymph node and ear. Interestingly, chrysin
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significantly inhibited the production of cytokines, Th2 chemokines, CCL17 and CCL22 by the down-regulation of p38 MAPK, NF-κB, and STAT1 in tumor necrosis factor (TNF)-α/interferon
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(IFN)-γ-stimulated human keratinocytes (HaCaT). Chrysin also inhibited TNF-α/IFN-γstimulated IL-33 expression in HaCaT cells and mouse primary keratinocytes. Taken together,
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the results indicate that chrysin suppressed AD symptoms, suggesting that chrysin might be a candidate for the treatment of AD and skin allergic diseases.
Key words: Chrysin; Atopic dermatitis; Keratinocytes; IL-33.
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Introduction Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by a type 2
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helper T cell (Th2) immune response and immunoglobulin (Ig) E sensitization (Leung et al., 2004). The pathogenesis of AD is multifactorial and involves a complex immunologic cascade, including skin barrier dysfunction, defects in the cutaneous cell-mediated immune response, genetic susceptibility factors, and environmental factors. Particularly, defects in epidermal skin
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barriers lead to elevated sensitivity to atopic aeroallergens including house dust mite (HDM). It recruits Th2 inflammatory cells to elicit the structural changes that occur in the skin of
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susceptible patients (Leung et al., 2004). In acute and chronic AD lesions, Th2-mediated immune responses involve many cell types, including eosinophils, basophils, mast cells, type 2 innate lymphoid cells (ILC2s), and their biological products (Hammad and Lambrecht, 2015). Th2, Th17, and Th22 cells are observed in both acute and chronic skin lesions, and are associated with
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inflamed skin, redness, itchy lesions, epidermal hyperplasia and lichenified chronic AD skin lesions (Dhingra and Guttman-Yassky, 2014; Gandhi et al., 2016). Th1 cells also can be detected in chronic lesions during AD development (Leung et al., 2004).
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Keratinocytes in the skin are the crucial providers or initiators of AD. Activated keratinocytes express various pro-inflammatory cytokines and chemokines such as tumor necrosis factor
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(TNF)-α, interleukin (IL)-1, IL-6, CCL17 and CCL22 which contribute to the inflammation in AD development (Bernard et al., 2012). Recently, it was demonstrated that keratinocytes recognize antigens in the presence of IL-33, subsequently leading to a feed forward loop that drives early Th2 and B cell immune responses, and a potentially later Th1 cell immune response in AD (Cevikbas and Steinhoff, 2012). Topical glucocorticoids and calcineurin inhibitors induce a rapid diminution in CCL17 levels,
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providing further evidence of an underlying Th2-driven response in AD therapies (Beck et al., 2014). Glucocorticoid use is reserved for patients with severe treatment-resistant AD (SCORAD ≥ 50% baseline, the initial dose, 2.5 and 5 mg/kg/day) (Cury Martins et al., 2015; Hatano et al.,
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2011; Langan et al., 2006; Mrowietz et al., 2009). Oral corticosteroids and calcineurin inhibitors improve the lesions of AD. However, long-term use of these drugs causes adverse effects such as telangiectatic lesions, cutaneous atrophy, steroid acne, and relapse (Coondoo et al., 2014; Cury
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Martins et al., 2015). Therefore, there is a need for more effective AD therapeutic agents with minimal negative side effects.
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Chrysin (5,7-dihydroxyflavone) is a flavonol-type flavonoid present in honey, bee propolis and many natural plant extracts (Sobocanec et al., 2006). Flavonoids have broad-acting therapeutic properties such as anti-allergic, anti-oxidative (Lapidot et al., 2002), anti-viral (Schnitzler et al., 2010), immunoregulatory (Zeinali et al., 2017) and anti-inflammatory actions
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(Dhawan et al., 2002). The common use of chrysin as a dietary supplement is to support healthy testosterone levels, aid cancer prevention, reduce oxidative stress through its anti-oxidant benefits, and support inflammation management (Feng et al., 2014; Gambelunghe et al., 2003;
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Kasala et al., 2016; Woo et al., 2005). Currently, no side effect of chrysin has been reported in the medical literature. Chrysin is a potential therapeutic agent for autoimmune diseases such as
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inflammatory bowel disease (Shin et al., 2009), multiple sclerosis (Zhang et al., 2015), neuritis (Xiao et al., 2014), and ocular inflammation (Meng et al., 2016). Despite interest in chrysin and its potential use in disease treatment, its mechanism and biological effects on AD are largely unknown. Thus, the aim of this study was to elucidate the effects of chrysin on AD, and to define the underlying mechanisms of these effects.
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Materials and methods Animals
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Six-week-old female BALB/c mice were purchased from SLC Inc. (Hamamatsu, Japan). The animals were housed with 5-10 mice per cage in a laminar air flow room maintained at a temperature of 22 ± 2°C with a relative humidity of 55 ± 5% throughout the study. The care and treatment of the mice were in accordance with the guidelines established by the Public Health
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Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the
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Institutional Animal Care and Use Committee.
Drugs and chemicals
Chrysin was purchased from Sigma (St. Louis, MO). Dermatophagoides farinae extract (DFE, Greer Laboratories, Lenoir, NC) was used as an antigen. All other reagents were purchased from
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Sigma unless otherwise stated. DFE was dissolved in phosphate-buffered saline containing 0.5% Tween 20. 2,4-dinitrochlorobenzene (DNCB, 1%) was dissolved in an acetone/olive oil (1:3) solution. Recombinant human TNF-α and IFN-γ were purchased from R&D systems
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(Minneapolis, MN).
Cell culture and viability
A human keratinocyte cell line, HaCaT, was maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/m penicillin G, 100 µg/ml streptomycin) at 37°C in 5% CO2. Cell viability was determined using the 3-(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide assay (MTT). After 24 h of treatment, MTT (5 mg/ml) was added into each well that contained a sample and incubated for 2 h. Isopropanol was added to dissolve the
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formazan crystals. Absorbance on each sample compared to that of the control, was calculated and expressed as a percentage.
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Induction of AD-like lesions in the mouse ear
The induction of AD-like lesions by DNCB and DFE was performed based on our previous research (Choi et al., 2013). A schematic experimental procedure is described in Figure 1A. Mice
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(n = 5/group) were randomly divided into four groups, and the surfaces of both ear lobes were stripped very gently with surgical tape (Nichiban, Tokyo, Japan). After stripping, 20 µl of DNCB
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(1%) was painted on each ear and then 20 µl of DFE (10 mg/ml) 4 days later. Treatment of DNCB/DFE was repeated once a week alternatively for 4 weeks. Two weeks after the first induction, tail bleeding was performed to check the serum IgE level. After confirming an atopic condition by IgE levels, the oral administration of chrysin (2, 10, and 50 mg/kg) was five times
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per week after day 7, for a total duration of the 4 weeks. The therapeutic drug for the treatment of AD, tacrolimus, was used as a positive control. Ear thickness was measured 24 h after DNCB or DFE application with a dial thickness gauge (Mitutoyo, Co., Tokyo, Japan). On day 28, blood
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samples were collected by celiac artery before euthanasia. The serum was stored at -70°C for further analysis. After blood collection, ears were removed and used for a histopathological
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analysis. Serum IgE level was measured using an ELISA kit (BD Biosciences, Oxford, UK) according to the manufacturer's instructions. The DFE-specific levels were indicated by the O.D. value.
Histological observation The ears were fixed with 10% formaldehyde and embedded in paraffin. Thin 5 µm sections were
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stained with hematoxylin and eosin. Infiltrated lymphocytes, thickening of the epidermis, and fibrosis in the dermis were observed by microscope. For measurement of mast cell infiltration, skin sections were stained with toluidine blue, and the number of mast cells in five sites chosen
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at random was counted. Eosinophils were counted blinded in 10 high-power fields at a magnification of 400×. Dermal thickness was analyzed in H&E-stained sections viewed under a magnification of 100×. Thickness was measured in five randomly selected fields from each
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Histamine assay
Histamine content was measured through the o-phthaldialdehyde spectrofluorometric procedure based on previous research (Je et al., 2015). The blood from the mice was centrifuged at 400 g for 10 min, and the serum was withdrawn to measure histamine content. Fluorescent intensity
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was measured using 355 nm excitation and 450 nm filters and the fluorescence Spectrometer LS50B (Perkin-Elmer, Norwalk, CT).
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FACS
At the end of the experiment, mice were euthanized and both auricular lymph node was collected
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from each mouse. Auricular lymph nodes were ground using 70 µm nylon cell strainers (Falcon, Bedford, MA) to isolate single cells. Cells were stained using a mouse CD4 PerCP-Cy™5.5FITC-conjugated Th1 (IFN-γ), CD4 PerCP-Cy™5.5-APC-conjugated Th2 (IL-4), and CD4 PerCP-Cy™5.5-PE-conjugated Th17 (IL-17A) phenotyping kit (BD Biosciences) according to the manufacturer's instructions. The fluorescence intensity was detected using a FACSCalibur flow cytometer (BD Biosciences).
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qPCR For the detection of expression of cytokines, qPCR was carried out using the Thermal Cycler
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Dice TP850 (Takarabio, Shiga, Japan) according to the manufacturer’s protocol. At the end of in vivo experimental period, the ears were excised, and total RNA was isolated. HaCaT cells were pretreated with chrysin for 1 h, and then stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml)
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for 6 h. Total cellular RNA was isolated from cells (2×105 cells/24-well plates) as described in previous research (Choi et al., 2013). Briefly, 2 µl of cDNA (100 ng), 1 µl of sense and antisense
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primer solution (0.4 µM), 12.5 µl of SYBR Premix Ex Taq (Takarabio), and 9.5 µl of dH2O were mixed together to obtain a final 25 µl reaction mixture in each reaction tube. The conditions for PCR were similar to ones as described in previous research (Choi et al., 2013) except for the primer of mouse IL-33 (F 5'-CTG GCC TCA CCA TAA GAA AGG AGA-3’, R 5'-AGG GAG
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GCA GGA GAC TGT GTT AAA-3’) and human IL-33 (F 5'-GGA GTG CTT TGC CTT TGG TA-3’, R 5'-TCA TTT GAG GGG TGT TGA GA-3’). The normalization and quantification of
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mRNA expression was performed using the TP850 software supplied by the manufacturer.
Nuclear protein extraction
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Samples for nuclear protein extraction were prepared as described in previous research (Choi et al., 2014). HaCaT cells were pretreated with chrysin for 1 h, and then stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 30 min. After stimulation, cells (2×106 cells/6-well plate) were washed in 1 ml of ice-cold PBS, centrifuged at 1,200 g for 5 min, resuspended in 400 µl of icecold hypotonic buffer (10 mM HEPES/KOH, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 10 min, vortexed, and centrifuged at 15,000 g for
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30 s. After washing, pelleted nuclei were resuspended in 50 µl of ice-cold saline buffer (50 mM HEPES/KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 20 min, vortexed, centrifuged at 15,000 g for 5 min at 4°C, and
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supernatant gathered.
Western blot
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Samples for Western blot were prepared as previously described (Choi et al., 2014). Briefly, cells (2×106 cells/6-well plate) were treated during 20 min to evaluate the activation of p38 MAPK
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and STAT1, and during 30 min to the activation of NF-κB. Cells were then rinsed twice with icecold PBS, and total cell lysates were gathered in 200 µl of lysis buffer. The lysates were spun in a micro-centrifuge for 20 min at 4°C, and the supernatant was collected. Proteins were electrophoresed using 8-12% SDS-PAGE, and then transferred to nitrocellulose membranes. The
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membranes were stained with reversible Ponceau S to ascertain equal loading of samples in the gel. Immunodetection was performed using an enhanced chemiluminescence detection kit
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(Amersham, Piscataway, NJ).
Mouse primary keratinocytes preparation
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Isolation and preparation were performed as following (Li et al., 2017). Keratinocytes were isolated from the tail skin of adult C57BL/6 mice (6-15 weeks). Tail skins were placed on 4 mg/ml dispase (Invitrogen, Carlsbad, CA) in keratinocyte growth medium (Invitrogen) overnight at 4°C. The next day, the epidermis was separated from the dermis, minced, and filtered with a 40-µm strainer. The isolated cells were seeded at a density of 1×104/cm2 in growth medium in culture dishes pre-coated with collagen. The medium was changed 24 h after the initial plating to
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removed unattached cells and harvest attached cells. For the experiment, isolated mouse primary keratinocytes were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 24 h.
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Statistical analysis
Statistical analyses were performed using Prism 7 (GraphPad Software, San Diego, CA). Treatment effects were analyzed using one way analysis of variance followed by Dunnett’s test.
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A value of p < 0.05 was used to indicate statistically significant differences.
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Results Chrysin inhibits the ear thickness and histopathological observation
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To identify the effect of chrysin on AD, we used the DNCB/DFE-induced AD mouse model. DNCB/DFE was topically applied on the ears of BALB/c mice; the oral administration of chrysin was five times per week after day 7, for a total duration of 28 days after the induction of AD-like skin inflammation (Fig. 1A). During the AD induction period, we measured ear
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thickness after 24 h of each DNCB or DFE application. The propensity of ear thickness of each group was similar until day 10. The oral administration of chrysin showed a significant decrease
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of ear thickness after 14 days (Fig. 1B). Tacrolimus (FK 506), a medication utilized clinically for the systemic therapy of AD, was used as a positive control.
Histological analysis of the ear skin showed many changes after AD induction, including inflammatory cell infiltration and hyperkeratosis (Fig. 2A), whereas chrysin was a significant
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ameliorator of AD-caused histologic changes. Epidermal and dermal thickness (Fig. 2B and C) as well as eosinophil infiltration (Fig. 2D) were also lowered. Activated mast cells release histamine which causing itching and inflammation in AD (Oyoshi et al., 2009). Thus, we
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examined mast cell infiltration and the serum levels of histamine. Chrysin administration
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resulted in reduced mast cell infiltration (Fig. 2E) and serum histamine levels (Fig. 3A).
Chrysin reduces the serum immunoglobulin and inflammatory cytokines Multiple AD mouse models have established a definite role for IL-4 in the pathogenesis of AD (Chan et al., 2001). These AD models demonstrated the typical pattern of early expression of Th2 cytokines, with a transition to a more Th1 phenotype with disease progression (Chen et al., 2004). IgE (Th2 phenotype) levels were increased in early stages of disease, with elevated levels
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of IgG2a (Th1 phenotype) presenting in later stages (Chen et al., 2005). To determine the ability of chrysin on the Th1 or Th2 responses, we assessed serum levels of IgE (total and DFE specific) and IgG2a. The serum of chrysin-treated mice exhibited significant reduction in levels of total
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IgE, DFE-specific IgE, and IgG2a compared to the AD group (Fig. 3B-D).
CD4+ T cells have been divided according to their effector cytokines. Beside the classic Th1 (TNF-α and IFN-γ), Th2 (IL-4, IL-13 and IL-31) and Th17 (IL-17A) subsets, a new subset has
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been defined as Th22 (IL-22) cells in human and mouse AD (Brandt and Sivaprasad, 2011). To analyze the role of chrysin in controlling Th responses, the effects of chrysin on CD4+ T cell
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subsets in the auricular lymph nodes were measured by FACS analysis 28 days after AD induction. The frequency of Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+) and Th17 (CD4+IL-17A+) cells were significantly increased in AD, but exhibited a marked diminution in chrysin treated mice (Fig. 4A-D). As expected, AD mice showed a high gene expression of various cytokines
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such as Th1 (TNF-α and IFN-γ), Th2 (IL-4, IL-13 and IL-31), Th17 (IL-17), and Th22 (IL-22); however chrysin treated mice showed a significant reduction in the expression of these cytokines
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(Fig. 5).
Chrysin suppresses the activation of keratinocytes
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Keratinocytes from patients with AD produce significantly higher concentrations of proinflammatory
cytokines
after
stimulation
with
TNF-α/IFN-γ (Leung
and
Bieber,
2003). Therefore, we used TNF-α/IFN-γ-stimulated keratinocytes to elucidate the biological activities and molecular mechanisms of chrysin. We first checked the cytotoxicity of chrysin. HaCaT was exposed to various concentrations of chrysin for 24 h. Chrysin did not show cytotoxicity upto 100 µg/ml (Fig. S1). Next, we investigate the effect of chrysin on the pro-
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inflammatory cytokines and Th2 chemokines in HaCaT cells and mouse primary keratinocytes. Chrysin suppressed TNF-α/IFN-γ-stimulated gene expression and/or protein levels of TNFα, IL-1β, IL-6, CCL17 and CCL22 in both human (Fig. 6A and B) and mouse keratinocytes (Fig.
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6C).
NF-κB, which is activated by MAPKs signaling, and STAT1 signaling pathways facilitate the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokines (CCL17
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and CCL22) in TNF-α/IFN-γ-stimulated HaCaT cells (Choi et al., 2013; Kwon et al., 2012).
NF-κB, and STAT1 pathways (Fig. 6D).
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Western blot analysis revealed that similar to the tacrolimus, chrysin suppressed the p38 MAPK,
Chrysin suppresses IL-33 in keratinocytes
Th2 inflammation, eosinophils, mast cells and basophils from AD have been shown to be sources
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of IL-33 (Akdis et al., 2011). In addition, IL-33 is one of the most crucial cytokines in driving increased Th1 and Th2 levels in skin diseases via keratinocytes (Balato et al., 2012; Balato et al., 2016). Therefore, we examined whether chrysin reduces the expression of IL-33 in keratinocytes.
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HaCaT cells were pretreated with chrysin for 1 h and stimulated with TNF-α/IFN-γ for 6 h.
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Interestingly, chrysin inhibited TNF-α/IFN-γ-stimulated gene expression and protein levels of IL-33 in HaCaT cells (Fig. 7A) and mouse primary keratinocytes (Fig. 7B). However, chrysin did not affect IL-33 in mouse ear tissue (Fig. S2). These results suggest that chrysin can decrease Th2 immune response in AD by regulating IL-33 secretion in keratinocytes. We confirmed the role of IL-33 using IL-33 siRNA. IL-33 siRNA was transfected before the TNF-α/IFN-γ stimulation in keratinocytes. IL-33 siRNA significantly reduced release of IL-1β and IL-6 in mouse primary keratinocytes. These results suggest that chrysin might suppress 13
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TNF-α/IFN-γ-activated secretion of pro-inflammatory cytokines by inhibiting IL-33 (Fig. 7C).
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Discussion In this study, we examined the effectiveness of chrysin on AD-like skin inflammation utilizing in vivo and in vitro models. AD is characterized by numerous inflammatory cell infiltrates,
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mainly comprising of CD4+ T cells situated in perivascular areas, as well as mast cells and eosinophil infiltrates in AD skin lesions, and an increase of epidermal and dermal thickness (Hammad and Lambrecht, 2015; Ong, 2014). The release of high amounts of histamine into the
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tissues via mast cells, and into the blood stream via basophils, occurs with repeated introduction of an allergic trigger over a period of time (Ohsawa and Hirasawa, 2014). Histamine release is
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associated with various symptoms such as heat, redness, swelling and itching (Buddenkotte et al., 2010). We showed that chrysin-treated mice exhibited decreased epidermal thickening in the ear, as well as diminished dermal infiltration of eosinophils and mast cells, similar to tacrolimus-treated mice. In addition, chrysin inhibited serum histamine and pathogenesis of the
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AD-like skin inflammation, a result that aligns with our previous study (Bae et al., 2011). Thus, we suggest that chrysin might be used to absolve pathogenic AD symptoms through the inhibition of histamine.
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Antigen specific-IgE contributes to the perpetuation of allergic inflammation, potentially through the promotion of IL-4 and/or IL-13 secreting activated Th2 cells (Galli and Tsai, 2012).
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This mechanism characterizes acute AD, along with activated skin-resident dendritic cells (Galli and Tsai, 2012). With the onset of acute AD, Th22 cells secrete IL-22, which induces epidermal hyperplasia and synergizes with the Th17 cytokine (Dhingra and Guttman-Yassky, 2014). Additionally, Th17 activity remains constant during acute disease (Bernard et al., 2012). In chronic lesions of AD, IgE producing Th2 cells and Th22 cells are present in skin infiltrates, as well as Th1 cells (Dhingra and Guttman-Yassky, 2014). Our data showed that chrysin reduced
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secretion of both total serum IgE and antigen specific IgE, and also Th1-derived IgG2a. Moreover, we found that chrysin suppressed Th1, Th2, Th17 and Th22 cell response in mouse lymph nodes and/or ears. Thus, chrysin may possess immune suppressive ability that can
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contribute to the treatment of whole AD stage.
Keratinocytes also regulate the release of cytokines, chemokines and proteases, thus contributing to the inflammatory reactions and immune responses in AD as part of the innate
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immune defense (Cevikbas and Steinhoff, 2012). TNF-α/IFN-γ-stimulated keratinocytes produce various pro-inflammatory cytokines, Th2 cell-attracting chemokines such as CCL17
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and CCL22, as well as innate pro-Th2 cytokines like IL-33 (Hammad and Lambrecht, 2015; Ito et al., 2005; Meephansan et al., 2012), which further enhance the infiltration of Th2 cells in AD lesions (Ong, 2014). In this study, we have shown that chrysin significantly decreased gene expression of CCL17 and CCL22 and the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6),
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similar to tacrolimus treatment. Previous studies showed that chrysin inhibits MAPKs in LPSinduced macrophages, and NF-κB in various cells (Bae et al., 2011; Lee et al., 2017; Woo et al., 2005). Concurrently, in our study, chrysin inhibited p38 MAPK, STAT1, as well as the
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degradation of IκBα and the nuclear translocation of NF-κB. These results demonstrate that
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chrysin can suppress CCL17 and CCL22-mediated Th2 infiltration by the down-regulation of p38 MAPK, NF-κB, and STAT1 signaling pathways in AD lesions. Unlike in humans where IL-33 possesses the semblance of an early cytokine in inflammatory response, mouse IL-33 levels remain relatively constant even during later stages of AD (Cevikbas and Steinhoff, 2012). Our results showed increased IL-33 in human keratinocytes and mouse ears during AD. Treatment with chrysin significantly reduced expression of IL-33 in human keratinocytes and mouse primary keratinocytes. However, chrysin did not affect the
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expression of IL-33 in mouse ears, although there was an overall reduction of AD-driven inflammation. This is presumably because we used a mixture of all skin components including keratinocytes, epidermis, and dermis.
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In response to exogenous factors, such as foreign pathogens, ultraviolet (UV) radiation and chemical irritants, innate immune cells specifically keratinocytes mount different types of responses including release of antimicrobial agents. Keratinocytes, therefore, as part of the
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innate immune defense, contribute to the inflammatory reactions and immune responses in AD by regulating the production of cytokines, chemokines, proteases, and bioactive lipids (Bangert
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et al., 2011; Gutowska-Owsiak and Ogg, 2012). Thus, upon stimulation by various allergens, toxins, or infectious agents, keratinocytes are capable of beginning a cross-talk between the innate and adaptive immune responses by stimulating T cells in patients with AD through the release of key mediators (Cevikbas and Steinhoff, 2012). On the other hand, the adaptive
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immune responses in the skin are initiated by dermal dendritic cells and epidermal dendritic antigen-presenting cells, and then executed by T lymphocytes (Bangert et al., 2011). Based on the presence of keratinocytes from the innate immune system in mouse ears, as well as various
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factors of the adaptive immune system, our findings suggest that chrysin may be more active in regulating AD through targeting the adaptive immune response. Additionally, chrysin can also
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be effective in suppressing the innate immune system in structural cells of the skin, such as keratinocytes, by inhibiting IL-33. In conclusion, chrysin is involved in orchestrating innate and adaptive inflammatory responses in the onset and perpetuation of AD but a more detailed future study should be conducted. Upon converting the dosage of mouse to human, chrysin can be available for oral administration with a maximum therapeutic concentration of 4.05 mg/kg/day in human AD.
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However, it is crucial to develop a safe and well-designed application for human AD. Nonetheless, we provide evidence that chrysin could be a potential therapeutic agent and dietary supplement for AD based on its inhibitory effects on skin inflammatory response, and may
Conflict of Interest
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The authors declare that there are no conflicts of interest.
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provide a novel approach to further understanding inflammatory skin diseases.
Acknowledgements
This study was supported by the National Research Foundation of Korea grant funded by the Korean Government (2014R1A5A2009242, 2016R1A2B4008513, 2017R1D1A1B03031032 and 2015R1D1A3A01016229), KRIBB Research Initiative Program (KGM4251723), and High
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Affairs.
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Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural
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Lapidot, T., Walker, M.D., Kanner, J., 2002. Antioxidant and prooxidant effects of phenolics on pancreatic beta-cells in vitro. J. Agric. Food Chem. 50, 7220-7225. Lee, B.K., Lee, W.J., Jung, Y.S., 2017. Chrysin attenuates VCAM-1 expression and monocyte
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Leung, D.Y., Bieber, T., 2003. Atopic dermatitis. Lancet. 361, 151-160. Leung, D.Y., Boguniewicz, M., Howell, M.D., Nomura, I., Hamid, Q.A., 2004. New insights into atopic dermatitis. J. Clin. Invest. 113, 651-657. Li, F., Adase, C.A., Zhang, L.J., 2017. Isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin. J. Vis. Exp. 14. Meephansan, J., Tsuda, H., Komine, M., Tominaga, S., Ohtsuki, M., 2012. Regulation of IL-33
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1-20 in mice. Cell. Mol. Immunol. 14, 702-711.
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mechanisms in atopic dermatitis. Adv. Immunol. 102, 135-226. Schnitzler, P., Neuner, A., Nolkemper, S., Zundel, C., Nowack, H., Sensch, K.H., Reichling, J., 2010. Antiviral activity and mode of action of propolis extracts and selected compounds.
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Sobocanec, S., Sverko, V., Balog, T., Saric, A., Rusak, G., Likic, S., Kusic, B., Katalinic, V., Radic, S., Marotti, T., 2006. Oxidant/antioxidant properties of Croatian native propolis. J. Agric. Food Chem. 54, 8018-8026. Woo, K.J., Jeong, Y.J., Inoue, H., Park, J.W., Kwon, T.K., 2005. Chrysin suppresses
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lipopolysaccharide-induced cyclooxygenase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity. FEBS Lett. 579, 705-711. Xiao, J., Zhai, H., Yao, Y., Wang, C., Jiang, W., Zhang, C., Simard, A.R., Zhang, R., Hao, J.,
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inflammatory properties of chrysin and flavonoids substances. Biomed. Pharmacother. 92, 998-1009.
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Zhang, K., Ge, Z., Xue, Z., Huang, W., Mei, M., Zhang, Q., Li, Y., Li, W., Zhang, Z., Zhang, L., Wang, H., Cai, J., Yao, Z., Zhang, R., Da, Y., 2015. Chrysin suppresses human CD14(+) monocyte-derived
dendritic
cells
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ameliorates
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encephalomyelitis. J. Neuroimmunol. 288, 13-20.
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Legends for Figures Figure 1. Experimental design and ear thickness in AD mice. (A) Experimental design for the induction of AD-like skin lesions. Mice (n = 5/group) were divided into four groups, and the
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surfaces of both ear lobes were stripped five times with surgical tape. After stripping, 20 µl of DNCB (1%) was painted on each ear and then 20 µl of DFE (10 mg/ml) 4 days later. Treatment with DNCB/DFE was repeated once a week alternatively for 4 weeks. Chrysin was orally
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administered (2, 10, or 50 mg/kg) for five times per week after day 7, for a total duration of the
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4 weeks. (B) Ear thickness was measured 24 h after DNCB or DFE application with a dial thickness gauge. Data are presented as mean ± SD of five determinations. *p < 0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac, tacrolimus.
Figure 2. Histological analysis of AD mice. (A) Representative photomicrographs of ear
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sections were stained with hematoxylin and eosin (upper panel, scale bar = 40 µm) or toluidine blue (lower panel, scale bar = 20 µm). (B, C) Epidermal and dermal thickness. (D, E) Numbers of eosinophils and mast cells. Data are presented as mean ± SD of five determinations. *p <
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0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac, tacrolimus.
Figure 3. Serum levels in AD mice. The blood samples of vehicle, DNCB/DFE plus vehicle, and DNCB/DFE plus chrysin (2, 10, or 50 mg/kg) groups were collected by celiac artery after 28 days. (A) Histamine levels were measured by histamine assay, and (B) Total serum IgE, (C) DFE-specific IgE, and (D) serum IgG2a levels were measured by ELISA. Data are presented as mean ± SD of five determinations. *p < 0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac, tacrolimus. 25
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Figure 4. Expansion of Th1, Th2, and Th17 cells in the lymph nodes during AD. (A) CD4+ T cells in lymph nodes of mice administered chrysin on day 28 after AD induction were analyzed
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by intracellular cytokine staining for FACS analysis. Numbers in quadrants indicate percentage of cells expressing CD4+ IFN-γ, IL-4, and/or IL-17. (B-D) Statistical analysis of IFN-γ, IL-4, or IL-17-expressing CD4+ T cells in the lymph nodes. Data are presented as mean ± SD of three
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determinations. *p < 0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac,
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tacrolimus.
Figure 5. Expression of cytokines in the ear of AD mice. To determine cytokine levels in AD mice, ears were excised on day 28. Gene expression was analyzed by qPCR. The gene expression levels were normalized to β-actin, and the values of fold-changes are represented.
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Data are presented as mean ± SD of five determinations. *p < 0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac, tacrolimus.
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Figure
inflammation
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TNF-α/IFN-γ-stimulated
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keratinocytes. (A) Gene expression of TNF-α, IL-1β, IL-6, CCL17, and CCL22 were determined by the qPCR. HaCaT cells were pretreated with chrysin (0.1, 1, or 10 µg/ml) 1 h before the stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 6 h. The gene expression levels were normalized to β-actin, and the values of fold-changes are represented. (B) Protein secretion of IL-1β, IL-6, CCL17, and CCL22 were determined by the ELISA. HaCaT cells were pretreated with chrysin (0.1, 1, or 10 µg/ml) 1 h before the stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 24 h. (C) Mouse primary keratinocytes were 26
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stimulated with TNF-α/IFN-γ for 24 h. The cells were then washed, cultured in serum-free medium with chrysin for 24 h. Culture supernatants were analyzed by ELISA. Data are
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presented as mean ± SD. *p < 0.05 significantly lower than TNF-α/IFN-γ-stimulation. (D) Inhibition of the p38 MAPK, NF-κB and STAT1 pathways. Cells were pretreated with chrysin (10 µg/ml) 1 h before stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 20 min. The experiment shown is representative of three independent experiments. N-NF-κB, nucleus NF-
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κB; Tac, tacrolimus.
Figure 7. Chrysin suppresses TNF-α/IFN-γ-stimulated IL-33 in keratinocytes. (A) HaCaT cells were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) during 6 h for gene expression and 24 h for protein secretion. The gene expression levels were normalized to β-actin, and the values of fold-changes are represented. (B) Mouse primary keratinocytes were stimulated with
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TNF-α/IFN-γ for 24 h. The cells were then washed, cultured in serum-free medium with chrysin for 24 h. Culture supernatants were analyzed by ELISA. (C) Analysis of IL-33 siRNA-
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transfected silencing of IL-1β and IL-6 production. Mouse primary keratinocytes were transiently transfected with IL-33 siRNA. After then, cells were stimulated with TNF-α/IFN-γ
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for 24 h. The cells were then washed, cultured in serum-free medium with chrysin for 24 h. Secretion of IL-1β and IL-6 was measured by ELISA. Data are presented as mean ± SD. *p < 0.05 significantly lower than TNF-α/IFN-γ-stimulation. Tac, tacrolimus.
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ACCEPTED MANUSCRIPT Highlights: • Chrysin suppresses AD symptoms by inhibiting T helper cell immune reaction.
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• Chrysin downregulates CCL17 and CCL22 expression via p38 MAPK, STAT1, and NFκB in human keratinocytes.
• Chrysin and tacrolimus have overlapping effects in inhibiting AD immune responses.
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• Chrysin might be a candidate for the treatment of skin allergic diseases.
S0278-6915(17)30623-3
DOI:
10.1016/j.fct.2017.10.025
Reference:
FCT 9350
To appear in:
Food and Chemical Toxicology
Received Date: 3 July 2017 Revised Date:
10 October 2017
Accepted Date: 13 October 2017
Please cite this article as: Choi, J.K., Jang, Y.H., Lee, S., Lee, S.-R., Choi, Y.-A., Jin, M., Choi, J.H., Park, J.H., Park, P.-H., Choi, H., Kwon, T.K., Khang, D., Kim, S.-H., Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes, Food and Chemical Toxicology (2017), doi: 10.1016/ j.fct.2017.10.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chrysin attenuates atopic dermatitis by suppressing inflammation of
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keratinocytes
Jin Kyeong Choi a, Yong Hyun Jang b, Soyoung Lee c, Sang-Rae Lee d, Young-Ae Choi a, Meiling Jin a, Jung Ho Choi e, Jee Hun Park e, Pil-Hoon Park f, Hyukjae Choi f, Taeg Kyu Kwon g,
Cell & Matrix Research Center, Department of Pharmacology, bDepartment of Dermatology,
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Dongwoo Khang h,*, Sang-Hyun Kim a,*
School of Medicine, Kyungpook National University, Daegu, Republic of Korea c
Immunoregulatory Materials Research Center, Jeongeup, dNational Primate Research Center,
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Republic of Korea R&D Center Pharmaceutical lab, Korean Drug Co., LTD, Seoul, Republic of Korea
f
College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea Department of Immunology, School of Medicine, Keimyung University, Daegu, Republic of
Korea
Department of Physiology, School of Medicine, Gachon University, Incheon, Republic of Korea
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Correspondence to: Prof. Sang-Hyun Kim, Department of Pharmacology, School of Medicine, Kyungpook National University, 101 Dongin-dong, Jung-gu, Daegu 700-422, Republic of Korea. E-mail: [email protected]; Tel: +82-53-420-4838; Fax: +82-53-423-4838 and Prof. Dongwoo Khang, Department of Physiology, School of Medicine, Gachon University, Incheon 406-840, Republic of Korea E-mail: [email protected]; Tel: +82-32-897-6515; Fax: +82-32-8996515. 1
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Abstract
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We previously reported the inhibitory effect of chrysin, a natural flavonoid plentifully contained in propolis, vegetables and fruits, on the mast cell-mediated allergic reaction. In this study, we evaluated the effect of chrysin on atopic dermatitis (AD) and defined underlying mechanisms of
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action. We used an AD model in BALB/c mice by the repeated local exposure of 2,4dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae extract, DFE) to
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the ears. Repeated alternative treatment of DNCB/DFE caused AD-like skin lesions. Oral administration of chrysin diminished AD symptoms such as ear thickness and histopathological analysis, in addition to serum IgE and IgG2a levels. Chrysin decreased infiltration of mast cells, and reduced serum histamine level. Chrysin also suppressed AD by inhibiting the inflammatory responses of Th1, Th2, and Th17 cells in mouse lymph node and ear. Interestingly, chrysin
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significantly inhibited the production of cytokines, Th2 chemokines, CCL17 and CCL22 by the down-regulation of p38 MAPK, NF-κB, and STAT1 in tumor necrosis factor (TNF)-α/interferon
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(IFN)-γ-stimulated human keratinocytes (HaCaT). Chrysin also inhibited TNF-α/IFN-γstimulated IL-33 expression in HaCaT cells and mouse primary keratinocytes. Taken together,
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the results indicate that chrysin suppressed AD symptoms, suggesting that chrysin might be a candidate for the treatment of AD and skin allergic diseases.
Key words: Chrysin; Atopic dermatitis; Keratinocytes; IL-33.
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Introduction Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by a type 2
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helper T cell (Th2) immune response and immunoglobulin (Ig) E sensitization (Leung et al., 2004). The pathogenesis of AD is multifactorial and involves a complex immunologic cascade, including skin barrier dysfunction, defects in the cutaneous cell-mediated immune response, genetic susceptibility factors, and environmental factors. Particularly, defects in epidermal skin
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barriers lead to elevated sensitivity to atopic aeroallergens including house dust mite (HDM). It recruits Th2 inflammatory cells to elicit the structural changes that occur in the skin of
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susceptible patients (Leung et al., 2004). In acute and chronic AD lesions, Th2-mediated immune responses involve many cell types, including eosinophils, basophils, mast cells, type 2 innate lymphoid cells (ILC2s), and their biological products (Hammad and Lambrecht, 2015). Th2, Th17, and Th22 cells are observed in both acute and chronic skin lesions, and are associated with
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inflamed skin, redness, itchy lesions, epidermal hyperplasia and lichenified chronic AD skin lesions (Dhingra and Guttman-Yassky, 2014; Gandhi et al., 2016). Th1 cells also can be detected in chronic lesions during AD development (Leung et al., 2004).
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Keratinocytes in the skin are the crucial providers or initiators of AD. Activated keratinocytes express various pro-inflammatory cytokines and chemokines such as tumor necrosis factor
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(TNF)-α, interleukin (IL)-1, IL-6, CCL17 and CCL22 which contribute to the inflammation in AD development (Bernard et al., 2012). Recently, it was demonstrated that keratinocytes recognize antigens in the presence of IL-33, subsequently leading to a feed forward loop that drives early Th2 and B cell immune responses, and a potentially later Th1 cell immune response in AD (Cevikbas and Steinhoff, 2012). Topical glucocorticoids and calcineurin inhibitors induce a rapid diminution in CCL17 levels,
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providing further evidence of an underlying Th2-driven response in AD therapies (Beck et al., 2014). Glucocorticoid use is reserved for patients with severe treatment-resistant AD (SCORAD ≥ 50% baseline, the initial dose, 2.5 and 5 mg/kg/day) (Cury Martins et al., 2015; Hatano et al.,
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2011; Langan et al., 2006; Mrowietz et al., 2009). Oral corticosteroids and calcineurin inhibitors improve the lesions of AD. However, long-term use of these drugs causes adverse effects such as telangiectatic lesions, cutaneous atrophy, steroid acne, and relapse (Coondoo et al., 2014; Cury
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Martins et al., 2015). Therefore, there is a need for more effective AD therapeutic agents with minimal negative side effects.
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Chrysin (5,7-dihydroxyflavone) is a flavonol-type flavonoid present in honey, bee propolis and many natural plant extracts (Sobocanec et al., 2006). Flavonoids have broad-acting therapeutic properties such as anti-allergic, anti-oxidative (Lapidot et al., 2002), anti-viral (Schnitzler et al., 2010), immunoregulatory (Zeinali et al., 2017) and anti-inflammatory actions
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(Dhawan et al., 2002). The common use of chrysin as a dietary supplement is to support healthy testosterone levels, aid cancer prevention, reduce oxidative stress through its anti-oxidant benefits, and support inflammation management (Feng et al., 2014; Gambelunghe et al., 2003;
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Kasala et al., 2016; Woo et al., 2005). Currently, no side effect of chrysin has been reported in the medical literature. Chrysin is a potential therapeutic agent for autoimmune diseases such as
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inflammatory bowel disease (Shin et al., 2009), multiple sclerosis (Zhang et al., 2015), neuritis (Xiao et al., 2014), and ocular inflammation (Meng et al., 2016). Despite interest in chrysin and its potential use in disease treatment, its mechanism and biological effects on AD are largely unknown. Thus, the aim of this study was to elucidate the effects of chrysin on AD, and to define the underlying mechanisms of these effects.
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Materials and methods Animals
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Six-week-old female BALB/c mice were purchased from SLC Inc. (Hamamatsu, Japan). The animals were housed with 5-10 mice per cage in a laminar air flow room maintained at a temperature of 22 ± 2°C with a relative humidity of 55 ± 5% throughout the study. The care and treatment of the mice were in accordance with the guidelines established by the Public Health
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Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the
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Institutional Animal Care and Use Committee.
Drugs and chemicals
Chrysin was purchased from Sigma (St. Louis, MO). Dermatophagoides farinae extract (DFE, Greer Laboratories, Lenoir, NC) was used as an antigen. All other reagents were purchased from
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Sigma unless otherwise stated. DFE was dissolved in phosphate-buffered saline containing 0.5% Tween 20. 2,4-dinitrochlorobenzene (DNCB, 1%) was dissolved in an acetone/olive oil (1:3) solution. Recombinant human TNF-α and IFN-γ were purchased from R&D systems
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(Minneapolis, MN).
Cell culture and viability
A human keratinocyte cell line, HaCaT, was maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/m penicillin G, 100 µg/ml streptomycin) at 37°C in 5% CO2. Cell viability was determined using the 3-(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide assay (MTT). After 24 h of treatment, MTT (5 mg/ml) was added into each well that contained a sample and incubated for 2 h. Isopropanol was added to dissolve the
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formazan crystals. Absorbance on each sample compared to that of the control, was calculated and expressed as a percentage.
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Induction of AD-like lesions in the mouse ear
The induction of AD-like lesions by DNCB and DFE was performed based on our previous research (Choi et al., 2013). A schematic experimental procedure is described in Figure 1A. Mice
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(n = 5/group) were randomly divided into four groups, and the surfaces of both ear lobes were stripped very gently with surgical tape (Nichiban, Tokyo, Japan). After stripping, 20 µl of DNCB
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(1%) was painted on each ear and then 20 µl of DFE (10 mg/ml) 4 days later. Treatment of DNCB/DFE was repeated once a week alternatively for 4 weeks. Two weeks after the first induction, tail bleeding was performed to check the serum IgE level. After confirming an atopic condition by IgE levels, the oral administration of chrysin (2, 10, and 50 mg/kg) was five times
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per week after day 7, for a total duration of the 4 weeks. The therapeutic drug for the treatment of AD, tacrolimus, was used as a positive control. Ear thickness was measured 24 h after DNCB or DFE application with a dial thickness gauge (Mitutoyo, Co., Tokyo, Japan). On day 28, blood
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samples were collected by celiac artery before euthanasia. The serum was stored at -70°C for further analysis. After blood collection, ears were removed and used for a histopathological
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analysis. Serum IgE level was measured using an ELISA kit (BD Biosciences, Oxford, UK) according to the manufacturer's instructions. The DFE-specific levels were indicated by the O.D. value.
Histological observation The ears were fixed with 10% formaldehyde and embedded in paraffin. Thin 5 µm sections were
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stained with hematoxylin and eosin. Infiltrated lymphocytes, thickening of the epidermis, and fibrosis in the dermis were observed by microscope. For measurement of mast cell infiltration, skin sections were stained with toluidine blue, and the number of mast cells in five sites chosen
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at random was counted. Eosinophils were counted blinded in 10 high-power fields at a magnification of 400×. Dermal thickness was analyzed in H&E-stained sections viewed under a magnification of 100×. Thickness was measured in five randomly selected fields from each
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Histamine assay
Histamine content was measured through the o-phthaldialdehyde spectrofluorometric procedure based on previous research (Je et al., 2015). The blood from the mice was centrifuged at 400 g for 10 min, and the serum was withdrawn to measure histamine content. Fluorescent intensity
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was measured using 355 nm excitation and 450 nm filters and the fluorescence Spectrometer LS50B (Perkin-Elmer, Norwalk, CT).
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FACS
At the end of the experiment, mice were euthanized and both auricular lymph node was collected
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from each mouse. Auricular lymph nodes were ground using 70 µm nylon cell strainers (Falcon, Bedford, MA) to isolate single cells. Cells were stained using a mouse CD4 PerCP-Cy™5.5FITC-conjugated Th1 (IFN-γ), CD4 PerCP-Cy™5.5-APC-conjugated Th2 (IL-4), and CD4 PerCP-Cy™5.5-PE-conjugated Th17 (IL-17A) phenotyping kit (BD Biosciences) according to the manufacturer's instructions. The fluorescence intensity was detected using a FACSCalibur flow cytometer (BD Biosciences).
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qPCR For the detection of expression of cytokines, qPCR was carried out using the Thermal Cycler
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Dice TP850 (Takarabio, Shiga, Japan) according to the manufacturer’s protocol. At the end of in vivo experimental period, the ears were excised, and total RNA was isolated. HaCaT cells were pretreated with chrysin for 1 h, and then stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml)
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for 6 h. Total cellular RNA was isolated from cells (2×105 cells/24-well plates) as described in previous research (Choi et al., 2013). Briefly, 2 µl of cDNA (100 ng), 1 µl of sense and antisense
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primer solution (0.4 µM), 12.5 µl of SYBR Premix Ex Taq (Takarabio), and 9.5 µl of dH2O were mixed together to obtain a final 25 µl reaction mixture in each reaction tube. The conditions for PCR were similar to ones as described in previous research (Choi et al., 2013) except for the primer of mouse IL-33 (F 5'-CTG GCC TCA CCA TAA GAA AGG AGA-3’, R 5'-AGG GAG
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GCA GGA GAC TGT GTT AAA-3’) and human IL-33 (F 5'-GGA GTG CTT TGC CTT TGG TA-3’, R 5'-TCA TTT GAG GGG TGT TGA GA-3’). The normalization and quantification of
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mRNA expression was performed using the TP850 software supplied by the manufacturer.
Nuclear protein extraction
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Samples for nuclear protein extraction were prepared as described in previous research (Choi et al., 2014). HaCaT cells were pretreated with chrysin for 1 h, and then stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 30 min. After stimulation, cells (2×106 cells/6-well plate) were washed in 1 ml of ice-cold PBS, centrifuged at 1,200 g for 5 min, resuspended in 400 µl of icecold hypotonic buffer (10 mM HEPES/KOH, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 10 min, vortexed, and centrifuged at 15,000 g for
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30 s. After washing, pelleted nuclei were resuspended in 50 µl of ice-cold saline buffer (50 mM HEPES/KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 20 min, vortexed, centrifuged at 15,000 g for 5 min at 4°C, and
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supernatant gathered.
Western blot
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Samples for Western blot were prepared as previously described (Choi et al., 2014). Briefly, cells (2×106 cells/6-well plate) were treated during 20 min to evaluate the activation of p38 MAPK
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and STAT1, and during 30 min to the activation of NF-κB. Cells were then rinsed twice with icecold PBS, and total cell lysates were gathered in 200 µl of lysis buffer. The lysates were spun in a micro-centrifuge for 20 min at 4°C, and the supernatant was collected. Proteins were electrophoresed using 8-12% SDS-PAGE, and then transferred to nitrocellulose membranes. The
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membranes were stained with reversible Ponceau S to ascertain equal loading of samples in the gel. Immunodetection was performed using an enhanced chemiluminescence detection kit
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(Amersham, Piscataway, NJ).
Mouse primary keratinocytes preparation
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Isolation and preparation were performed as following (Li et al., 2017). Keratinocytes were isolated from the tail skin of adult C57BL/6 mice (6-15 weeks). Tail skins were placed on 4 mg/ml dispase (Invitrogen, Carlsbad, CA) in keratinocyte growth medium (Invitrogen) overnight at 4°C. The next day, the epidermis was separated from the dermis, minced, and filtered with a 40-µm strainer. The isolated cells were seeded at a density of 1×104/cm2 in growth medium in culture dishes pre-coated with collagen. The medium was changed 24 h after the initial plating to
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removed unattached cells and harvest attached cells. For the experiment, isolated mouse primary keratinocytes were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 24 h.
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Statistical analysis
Statistical analyses were performed using Prism 7 (GraphPad Software, San Diego, CA). Treatment effects were analyzed using one way analysis of variance followed by Dunnett’s test.
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A value of p < 0.05 was used to indicate statistically significant differences.
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Results Chrysin inhibits the ear thickness and histopathological observation
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To identify the effect of chrysin on AD, we used the DNCB/DFE-induced AD mouse model. DNCB/DFE was topically applied on the ears of BALB/c mice; the oral administration of chrysin was five times per week after day 7, for a total duration of 28 days after the induction of AD-like skin inflammation (Fig. 1A). During the AD induction period, we measured ear
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thickness after 24 h of each DNCB or DFE application. The propensity of ear thickness of each group was similar until day 10. The oral administration of chrysin showed a significant decrease
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of ear thickness after 14 days (Fig. 1B). Tacrolimus (FK 506), a medication utilized clinically for the systemic therapy of AD, was used as a positive control.
Histological analysis of the ear skin showed many changes after AD induction, including inflammatory cell infiltration and hyperkeratosis (Fig. 2A), whereas chrysin was a significant
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ameliorator of AD-caused histologic changes. Epidermal and dermal thickness (Fig. 2B and C) as well as eosinophil infiltration (Fig. 2D) were also lowered. Activated mast cells release histamine which causing itching and inflammation in AD (Oyoshi et al., 2009). Thus, we
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examined mast cell infiltration and the serum levels of histamine. Chrysin administration
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resulted in reduced mast cell infiltration (Fig. 2E) and serum histamine levels (Fig. 3A).
Chrysin reduces the serum immunoglobulin and inflammatory cytokines Multiple AD mouse models have established a definite role for IL-4 in the pathogenesis of AD (Chan et al., 2001). These AD models demonstrated the typical pattern of early expression of Th2 cytokines, with a transition to a more Th1 phenotype with disease progression (Chen et al., 2004). IgE (Th2 phenotype) levels were increased in early stages of disease, with elevated levels
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of IgG2a (Th1 phenotype) presenting in later stages (Chen et al., 2005). To determine the ability of chrysin on the Th1 or Th2 responses, we assessed serum levels of IgE (total and DFE specific) and IgG2a. The serum of chrysin-treated mice exhibited significant reduction in levels of total
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IgE, DFE-specific IgE, and IgG2a compared to the AD group (Fig. 3B-D).
CD4+ T cells have been divided according to their effector cytokines. Beside the classic Th1 (TNF-α and IFN-γ), Th2 (IL-4, IL-13 and IL-31) and Th17 (IL-17A) subsets, a new subset has
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been defined as Th22 (IL-22) cells in human and mouse AD (Brandt and Sivaprasad, 2011). To analyze the role of chrysin in controlling Th responses, the effects of chrysin on CD4+ T cell
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subsets in the auricular lymph nodes were measured by FACS analysis 28 days after AD induction. The frequency of Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+) and Th17 (CD4+IL-17A+) cells were significantly increased in AD, but exhibited a marked diminution in chrysin treated mice (Fig. 4A-D). As expected, AD mice showed a high gene expression of various cytokines
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such as Th1 (TNF-α and IFN-γ), Th2 (IL-4, IL-13 and IL-31), Th17 (IL-17), and Th22 (IL-22); however chrysin treated mice showed a significant reduction in the expression of these cytokines
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(Fig. 5).
Chrysin suppresses the activation of keratinocytes
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Keratinocytes from patients with AD produce significantly higher concentrations of proinflammatory
cytokines
after
stimulation
with
TNF-α/IFN-γ (Leung
and
Bieber,
2003). Therefore, we used TNF-α/IFN-γ-stimulated keratinocytes to elucidate the biological activities and molecular mechanisms of chrysin. We first checked the cytotoxicity of chrysin. HaCaT was exposed to various concentrations of chrysin for 24 h. Chrysin did not show cytotoxicity upto 100 µg/ml (Fig. S1). Next, we investigate the effect of chrysin on the pro-
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inflammatory cytokines and Th2 chemokines in HaCaT cells and mouse primary keratinocytes. Chrysin suppressed TNF-α/IFN-γ-stimulated gene expression and/or protein levels of TNFα, IL-1β, IL-6, CCL17 and CCL22 in both human (Fig. 6A and B) and mouse keratinocytes (Fig.
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6C).
NF-κB, which is activated by MAPKs signaling, and STAT1 signaling pathways facilitate the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokines (CCL17
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and CCL22) in TNF-α/IFN-γ-stimulated HaCaT cells (Choi et al., 2013; Kwon et al., 2012).
NF-κB, and STAT1 pathways (Fig. 6D).
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Western blot analysis revealed that similar to the tacrolimus, chrysin suppressed the p38 MAPK,
Chrysin suppresses IL-33 in keratinocytes
Th2 inflammation, eosinophils, mast cells and basophils from AD have been shown to be sources
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of IL-33 (Akdis et al., 2011). In addition, IL-33 is one of the most crucial cytokines in driving increased Th1 and Th2 levels in skin diseases via keratinocytes (Balato et al., 2012; Balato et al., 2016). Therefore, we examined whether chrysin reduces the expression of IL-33 in keratinocytes.
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HaCaT cells were pretreated with chrysin for 1 h and stimulated with TNF-α/IFN-γ for 6 h.
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Interestingly, chrysin inhibited TNF-α/IFN-γ-stimulated gene expression and protein levels of IL-33 in HaCaT cells (Fig. 7A) and mouse primary keratinocytes (Fig. 7B). However, chrysin did not affect IL-33 in mouse ear tissue (Fig. S2). These results suggest that chrysin can decrease Th2 immune response in AD by regulating IL-33 secretion in keratinocytes. We confirmed the role of IL-33 using IL-33 siRNA. IL-33 siRNA was transfected before the TNF-α/IFN-γ stimulation in keratinocytes. IL-33 siRNA significantly reduced release of IL-1β and IL-6 in mouse primary keratinocytes. These results suggest that chrysin might suppress 13
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TNF-α/IFN-γ-activated secretion of pro-inflammatory cytokines by inhibiting IL-33 (Fig. 7C).
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Discussion In this study, we examined the effectiveness of chrysin on AD-like skin inflammation utilizing in vivo and in vitro models. AD is characterized by numerous inflammatory cell infiltrates,
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mainly comprising of CD4+ T cells situated in perivascular areas, as well as mast cells and eosinophil infiltrates in AD skin lesions, and an increase of epidermal and dermal thickness (Hammad and Lambrecht, 2015; Ong, 2014). The release of high amounts of histamine into the
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tissues via mast cells, and into the blood stream via basophils, occurs with repeated introduction of an allergic trigger over a period of time (Ohsawa and Hirasawa, 2014). Histamine release is
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associated with various symptoms such as heat, redness, swelling and itching (Buddenkotte et al., 2010). We showed that chrysin-treated mice exhibited decreased epidermal thickening in the ear, as well as diminished dermal infiltration of eosinophils and mast cells, similar to tacrolimus-treated mice. In addition, chrysin inhibited serum histamine and pathogenesis of the
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AD-like skin inflammation, a result that aligns with our previous study (Bae et al., 2011). Thus, we suggest that chrysin might be used to absolve pathogenic AD symptoms through the inhibition of histamine.
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Antigen specific-IgE contributes to the perpetuation of allergic inflammation, potentially through the promotion of IL-4 and/or IL-13 secreting activated Th2 cells (Galli and Tsai, 2012).
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This mechanism characterizes acute AD, along with activated skin-resident dendritic cells (Galli and Tsai, 2012). With the onset of acute AD, Th22 cells secrete IL-22, which induces epidermal hyperplasia and synergizes with the Th17 cytokine (Dhingra and Guttman-Yassky, 2014). Additionally, Th17 activity remains constant during acute disease (Bernard et al., 2012). In chronic lesions of AD, IgE producing Th2 cells and Th22 cells are present in skin infiltrates, as well as Th1 cells (Dhingra and Guttman-Yassky, 2014). Our data showed that chrysin reduced
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secretion of both total serum IgE and antigen specific IgE, and also Th1-derived IgG2a. Moreover, we found that chrysin suppressed Th1, Th2, Th17 and Th22 cell response in mouse lymph nodes and/or ears. Thus, chrysin may possess immune suppressive ability that can
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contribute to the treatment of whole AD stage.
Keratinocytes also regulate the release of cytokines, chemokines and proteases, thus contributing to the inflammatory reactions and immune responses in AD as part of the innate
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immune defense (Cevikbas and Steinhoff, 2012). TNF-α/IFN-γ-stimulated keratinocytes produce various pro-inflammatory cytokines, Th2 cell-attracting chemokines such as CCL17
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and CCL22, as well as innate pro-Th2 cytokines like IL-33 (Hammad and Lambrecht, 2015; Ito et al., 2005; Meephansan et al., 2012), which further enhance the infiltration of Th2 cells in AD lesions (Ong, 2014). In this study, we have shown that chrysin significantly decreased gene expression of CCL17 and CCL22 and the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6),
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similar to tacrolimus treatment. Previous studies showed that chrysin inhibits MAPKs in LPSinduced macrophages, and NF-κB in various cells (Bae et al., 2011; Lee et al., 2017; Woo et al., 2005). Concurrently, in our study, chrysin inhibited p38 MAPK, STAT1, as well as the
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degradation of IκBα and the nuclear translocation of NF-κB. These results demonstrate that
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chrysin can suppress CCL17 and CCL22-mediated Th2 infiltration by the down-regulation of p38 MAPK, NF-κB, and STAT1 signaling pathways in AD lesions. Unlike in humans where IL-33 possesses the semblance of an early cytokine in inflammatory response, mouse IL-33 levels remain relatively constant even during later stages of AD (Cevikbas and Steinhoff, 2012). Our results showed increased IL-33 in human keratinocytes and mouse ears during AD. Treatment with chrysin significantly reduced expression of IL-33 in human keratinocytes and mouse primary keratinocytes. However, chrysin did not affect the
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expression of IL-33 in mouse ears, although there was an overall reduction of AD-driven inflammation. This is presumably because we used a mixture of all skin components including keratinocytes, epidermis, and dermis.
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In response to exogenous factors, such as foreign pathogens, ultraviolet (UV) radiation and chemical irritants, innate immune cells specifically keratinocytes mount different types of responses including release of antimicrobial agents. Keratinocytes, therefore, as part of the
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innate immune defense, contribute to the inflammatory reactions and immune responses in AD by regulating the production of cytokines, chemokines, proteases, and bioactive lipids (Bangert
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et al., 2011; Gutowska-Owsiak and Ogg, 2012). Thus, upon stimulation by various allergens, toxins, or infectious agents, keratinocytes are capable of beginning a cross-talk between the innate and adaptive immune responses by stimulating T cells in patients with AD through the release of key mediators (Cevikbas and Steinhoff, 2012). On the other hand, the adaptive
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immune responses in the skin are initiated by dermal dendritic cells and epidermal dendritic antigen-presenting cells, and then executed by T lymphocytes (Bangert et al., 2011). Based on the presence of keratinocytes from the innate immune system in mouse ears, as well as various
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factors of the adaptive immune system, our findings suggest that chrysin may be more active in regulating AD through targeting the adaptive immune response. Additionally, chrysin can also
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be effective in suppressing the innate immune system in structural cells of the skin, such as keratinocytes, by inhibiting IL-33. In conclusion, chrysin is involved in orchestrating innate and adaptive inflammatory responses in the onset and perpetuation of AD but a more detailed future study should be conducted. Upon converting the dosage of mouse to human, chrysin can be available for oral administration with a maximum therapeutic concentration of 4.05 mg/kg/day in human AD.
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However, it is crucial to develop a safe and well-designed application for human AD. Nonetheless, we provide evidence that chrysin could be a potential therapeutic agent and dietary supplement for AD based on its inhibitory effects on skin inflammatory response, and may
Conflict of Interest
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The authors declare that there are no conflicts of interest.
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provide a novel approach to further understanding inflammatory skin diseases.
Acknowledgements
This study was supported by the National Research Foundation of Korea grant funded by the Korean Government (2014R1A5A2009242, 2016R1A2B4008513, 2017R1D1A1B03031032 and 2015R1D1A3A01016229), KRIBB Research Initiative Program (KGM4251723), and High
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Affairs.
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Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural
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mechanisms in atopic dermatitis. Adv. Immunol. 102, 135-226. Schnitzler, P., Neuner, A., Nolkemper, S., Zundel, C., Nowack, H., Sensch, K.H., Reichling, J., 2010. Antiviral activity and mode of action of propolis extracts and selected compounds.
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Phytother. Res. 24 Suppl 1, S20-28.
Shin, E.K., Kwon, H.S., Kim, Y.H., Shin, H.K., Kim, J.K., 2009. Chrysin, a natural flavone,
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improves murine inflammatory bowel diseases. Biochem. Biophys. Res. Commun. 381, 502-507.
Sobocanec, S., Sverko, V., Balog, T., Saric, A., Rusak, G., Likic, S., Kusic, B., Katalinic, V., Radic, S., Marotti, T., 2006. Oxidant/antioxidant properties of Croatian native propolis. J. Agric. Food Chem. 54, 8018-8026. Woo, K.J., Jeong, Y.J., Inoue, H., Park, J.W., Kwon, T.K., 2005. Chrysin suppresses
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lipopolysaccharide-induced cyclooxygenase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity. FEBS Lett. 579, 705-711. Xiao, J., Zhai, H., Yao, Y., Wang, C., Jiang, W., Zhang, C., Simard, A.R., Zhang, R., Hao, J.,
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2014. Chrysin attenuates experimental autoimmune neuritis by suppressing immunoinflammatory responses. Neuroscience 262, 156-164.
Zeinali, M., Rezaee, S.A., Hosseinzadeh, H., 2017. An overview on immunoregulatory and anti-
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inflammatory properties of chrysin and flavonoids substances. Biomed. Pharmacother. 92, 998-1009.
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Zhang, K., Ge, Z., Xue, Z., Huang, W., Mei, M., Zhang, Q., Li, Y., Li, W., Zhang, Z., Zhang, L., Wang, H., Cai, J., Yao, Z., Zhang, R., Da, Y., 2015. Chrysin suppresses human CD14(+) monocyte-derived
dendritic
cells
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ameliorates
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encephalomyelitis. J. Neuroimmunol. 288, 13-20.
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Legends for Figures Figure 1. Experimental design and ear thickness in AD mice. (A) Experimental design for the induction of AD-like skin lesions. Mice (n = 5/group) were divided into four groups, and the
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surfaces of both ear lobes were stripped five times with surgical tape. After stripping, 20 µl of DNCB (1%) was painted on each ear and then 20 µl of DFE (10 mg/ml) 4 days later. Treatment with DNCB/DFE was repeated once a week alternatively for 4 weeks. Chrysin was orally
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4 weeks. (B) Ear thickness was measured 24 h after DNCB or DFE application with a dial thickness gauge. Data are presented as mean ± SD of five determinations. *p < 0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac, tacrolimus.
Figure 2. Histological analysis of AD mice. (A) Representative photomicrographs of ear
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sections were stained with hematoxylin and eosin (upper panel, scale bar = 40 µm) or toluidine blue (lower panel, scale bar = 20 µm). (B, C) Epidermal and dermal thickness. (D, E) Numbers of eosinophils and mast cells. Data are presented as mean ± SD of five determinations. *p <
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Figure 3. Serum levels in AD mice. The blood samples of vehicle, DNCB/DFE plus vehicle, and DNCB/DFE plus chrysin (2, 10, or 50 mg/kg) groups were collected by celiac artery after 28 days. (A) Histamine levels were measured by histamine assay, and (B) Total serum IgE, (C) DFE-specific IgE, and (D) serum IgG2a levels were measured by ELISA. Data are presented as mean ± SD of five determinations. *p < 0.05 significantly lower than AD. Ctrl, control; AD, atopic dermatitis; Tac, tacrolimus. 25
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Figure 4. Expansion of Th1, Th2, and Th17 cells in the lymph nodes during AD. (A) CD4+ T cells in lymph nodes of mice administered chrysin on day 28 after AD induction were analyzed
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by intracellular cytokine staining for FACS analysis. Numbers in quadrants indicate percentage of cells expressing CD4+ IFN-γ, IL-4, and/or IL-17. (B-D) Statistical analysis of IFN-γ, IL-4, or IL-17-expressing CD4+ T cells in the lymph nodes. Data are presented as mean ± SD of three
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Figure 5. Expression of cytokines in the ear of AD mice. To determine cytokine levels in AD mice, ears were excised on day 28. Gene expression was analyzed by qPCR. The gene expression levels were normalized to β-actin, and the values of fold-changes are represented.
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Chrysin
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Th2-mediated
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inflammation
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TNF-α/IFN-γ-stimulated
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keratinocytes. (A) Gene expression of TNF-α, IL-1β, IL-6, CCL17, and CCL22 were determined by the qPCR. HaCaT cells were pretreated with chrysin (0.1, 1, or 10 µg/ml) 1 h before the stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 6 h. The gene expression levels were normalized to β-actin, and the values of fold-changes are represented. (B) Protein secretion of IL-1β, IL-6, CCL17, and CCL22 were determined by the ELISA. HaCaT cells were pretreated with chrysin (0.1, 1, or 10 µg/ml) 1 h before the stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 24 h. (C) Mouse primary keratinocytes were 26
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stimulated with TNF-α/IFN-γ for 24 h. The cells were then washed, cultured in serum-free medium with chrysin for 24 h. Culture supernatants were analyzed by ELISA. Data are
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presented as mean ± SD. *p < 0.05 significantly lower than TNF-α/IFN-γ-stimulation. (D) Inhibition of the p38 MAPK, NF-κB and STAT1 pathways. Cells were pretreated with chrysin (10 µg/ml) 1 h before stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 20 min. The experiment shown is representative of three independent experiments. N-NF-κB, nucleus NF-
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Figure 7. Chrysin suppresses TNF-α/IFN-γ-stimulated IL-33 in keratinocytes. (A) HaCaT cells were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) during 6 h for gene expression and 24 h for protein secretion. The gene expression levels were normalized to β-actin, and the values of fold-changes are represented. (B) Mouse primary keratinocytes were stimulated with
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TNF-α/IFN-γ for 24 h. The cells were then washed, cultured in serum-free medium with chrysin for 24 h. Culture supernatants were analyzed by ELISA. (C) Analysis of IL-33 siRNA-
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transfected silencing of IL-1β and IL-6 production. Mouse primary keratinocytes were transiently transfected with IL-33 siRNA. After then, cells were stimulated with TNF-α/IFN-γ
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for 24 h. The cells were then washed, cultured in serum-free medium with chrysin for 24 h. Secretion of IL-1β and IL-6 was measured by ELISA. Data are presented as mean ± SD. *p < 0.05 significantly lower than TNF-α/IFN-γ-stimulation. Tac, tacrolimus.
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ACCEPTED MANUSCRIPT Highlights: • Chrysin suppresses AD symptoms by inhibiting T helper cell immune reaction.
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• Chrysin downregulates CCL17 and CCL22 expression via p38 MAPK, STAT1, and NFκB in human keratinocytes.
• Chrysin and tacrolimus have overlapping effects in inhibiting AD immune responses.
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• Chrysin might be a candidate for the treatment of skin allergic diseases.