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Esculetin from Fraxinus rhynchophylla attenuates atopic skin inflammation by inhibiting the expression of inflammatory cytokines
International Immunopharmacology 59 (2018) 209–216
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Esculetin from Fraxinus rhynchophylla attenuates atopic skin inflammation by inhibiting the expression of inflammatory cytokines
T
Na-Hee Jeonga,1, Eun-Ju Yangb,1, Meiling Jina, Jong Yeong Leea, Young-Ae Choia, Pil-Hoon Parkc, ⁎ Sang-Rae Leed, Sun-Uk Kimd, Tae-Yong Shine, Taeg Kyu Kwonf, Yong Hyun Jangg, , ⁎⁎ ⁎⁎⁎ Kyung-Sik Songb, , Sang-Hyun Kima, a
Cell & Matrix Research Institute, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea c College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea d National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea e College of Pharmacy, Woosuk University, Jeonju, Republic of Korea f Department of Immunology and School of Medicine, Keimyung University, Daegu, Republic of Korea g Department of Dermatology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Atopic dermatitis Esculetin Keratinocytes House dust mite
Atopic dermatitis (AD) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. We aimed to investigate the effects of esculetin from Fraxinus rhynchophylla on atopic skin inflammation. For induction of atopic skin inflammation, we exposed the ears of female BALB/c mice to house dust mite (Dermatophagoides farinae extract, DFE) and 2,4-dinitrochlorobenzene (DNCB) for 4 weeks. Oral administration of esculetin reduced the symptoms of DFE/DNCB-induced atopic skin inflammation, which were evaluated based on ear swelling and number of scratch bouts. The immunoglobulin (Ig) E, IgG2a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. It suppressed production of Th1, Th2 and Th17-related cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, IL-13, IL-31 and IL-17 in the ear tissue. Furthermore, we investigated the effects of esculetin on activated keratinocytes, which are representative cells used for studying the pathogenesis of acute and chronic atopic skin inflammation. As results, esculetin suppressed gene expression of Th1, Th2 and Th17 cytokines and the activation of nuclear factor-κB and signal transducer and activator of transcription 1 in TNF-α/IFN-γ-stimulated keratinocytes. Taken together, these results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin could be a potential therapeutic candidate for the treatment of AD.
1. Introduction Atopic dermatitis (AD) is a well-known allergic disorder and a common chronic inflammation disease characterized by severs, prolonged itching, lichenification, papules, excoriations and recurrent eczematous lesions [1]. AD affects millions of children and adults worldwide, which often reduces the quality of life of patients markedly. The pathogenesis of AD seems to be caused by a complex interaction between the immune system and skin barrier [2]. The pathogenesis of AD involves T helper (Th) cell immune
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responses, and Th1 and Th2 cytokines have been reported to play an important role in skin inflammation specifically AD [3]. AD is classified into two phases: acute and chronic AD. In the acute phase, Th2 and Th17 cells are mainly activated, whereas in the chronic phase, Th1 cells are mainly activated [4]. A representative Th2 cytokine interleukin (IL)-4 is an activator and driver of AD, and initiates immunoglobulin (Ig) E isotype class switching from B cells [5]. Recently, elevated levels of IL-17A produced by Th17 cells were reported in skin lesions of patients with AD, which in turn led to the release of pro-inflammatory cytokines and chemokines [6]. Then, typical Th1 cytokine interferon
Corresponding author at: Department of Dermatology, School of Medicine, Kyungpook National University, #680, Gukchaebosang-ro, Jung-gu, Daegu 41944, Republic of Korea. Corresponding author at: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Daegu 41566, Republic of Korea. Corresponding author at: Department of Pharmacology, School of Medicine, Kyungpook National University, #680, Gukchaebosang-ro, Jung-gu, Daegu 41944, Republic of Korea. E-mail addresses: [email protected] (Y.H. Jang), [email protected] (K.-S. Song), [email protected] (S.-H. Kim). 1 These authors contributed equally to this work. ⁎⁎
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https://doi.org/10.1016/j.intimp.2018.04.005 Received 16 November 2017; Received in revised form 1 March 2018; Accepted 4 April 2018 1567-5769/ © 2018 Elsevier B.V. All rights reserved.
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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 mice were provided ad libitum access to food and water, and kept on a 12 h light:dark cycle. All care and treatment of the mice were in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kyungpook National University.
(IFN)-γ contributes to the infiltration of inflammatory cells that exacerbate chronic AD and leads to the production of IgG2a [7,8]. The most commonly used therapeutic agents for AD are topical corticosteroids, immunosuppressants, anti-histamines, anti-biotics, and calcineurin inhibitors. They rapidly suppress clinical responses safely and effectively in patients with AD and are cost effective [9]. However, continuous application of corticosteroids and immunosuppressants causes serious adverse effects, such as skin atrophy or thinning, contact dermatitis, telangiectases, purpura, and striae formation [10]. Therefore, there has been an increasing focus on natural therapeutic agents as alternative medicines, because they can provide an immunomodulatory effects with fewer side effects [11]. Esculetin (also known as aesculetin, 6,7-dihydroxycoumarin or cichorigenin), a derivative of coumarin, is a phenolic compound and exists in many plants known to have medicinal effects, such as Artemisia capillaris (Compositae), Citrus limonia (Rutaceae), Ceratostigma willmottianum (Plumbaginaceae), and Fraxinus rhynchophylla (Oleaceae) [12–14]. Esculetin has been known to exhibit anti-inflammatory activity in acute lung injury, anti-nociceptive effect in inflammatory pain, anti-oxidative effect in human colon cancer, anti-cancer effect in human gastric cancer, and effectiveness against allergic asthma [13,15–18]. In this study, we investigated the pharmacological effects of esculetin isolated from F. rhynchophylla on Dermatophagoides farinae extract (DFE) and 2,4-dinitrochlorobenzene (DNCB)-induced atopic skin inflammation in a mouse model and in keratinocytes.
2.3. Chemicals DFE (Greer Laboratories, Lenoir, NC) and DNCB were used as antigen and hapten respectively, for the induction of atopic skin inflammation. All other reagents were purchased from Sigma-Aldrich unless otherwise stated. DFE was dissolved in phosphate-buffered saline (PBS) containing 0.5% Tween 20. DNCB (1%) was dissolved in an acetone/olive oil (1:3) solution. Antibiotics, and trypsin-EDTA were obtained from Invitrogen (Grand Island, NY). Recombinant tumor necrosis factor (TNF)-α and IFN-γ were purchased from R&D systems (Minneapolis, MN). 2.4. Cell culture Human HaCaT keratinocytes were maintained in Dulbecco's Modified Eagle's Medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and antibiotics (100 U/mL penicillin G, 100 μg/mL streptomycin) at 37 °C in 5% carbon dioxide (CO2). Passages 3 to 6 were used throughout the study.
2. Materials and methods 2.1. Extraction, isolation, and identification
2.5. Induction of atopic skin inflammation in mouse ear The dried stem barks (4 kg) of F. rhynchophylla purchased from the local market (Daegu, Republic of Korea), were refluxed twice with 95% ethanol (EtOH) for 2 h. The extracted solution was filtered through a filter paper (Advantec, Tokyo, Japan), and the filtrate was concentrated to dryness by a rotary evaporator (EYELA, Tokyo, Japan) under reduced pressure below 40 °C. The EtOH extract (510 g) was suspended in distilled water, and successively partitioned with dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). The EtOAcsoluble fraction (45 g) was applied to a silica gel (70–230 mesh, Merck, Darmstadt, Germany) column [Ø 5.5 × 83 cm, chloroform (CHCl3): methanol (MeOH) = 20:1–11] to obtain nine fractions (Fr. 1-9). Fr. 5 was rechromatographed by using silica gel (70–230 mesh, Ø 2.5 × 83 cm, CH2Cl2: MeOH = 20:1-1:1) to yield six subfractions (Fr. 51 to 5-6). Compound 1 (20 mg) was isolated as an amorphous yellow powder via silica gel column chromatography (230–400 mesh, Ø 1 × 60 cm, EtOAc:MeOH:H2O = 100:1:1% - 10:1:1%) from Fr. 5-4. To identify the isolated compound 1, nuclear magnetic resonance (NMR; Bruker Avance Digital 500 NMR spectrometer, Karlsruhe, Germany) analysis was performed. Compound 1 was dissolved in deuterated methanol (CD3OD, Sigma-Aldrich, St. Louis, MO) before NMR analysis, and tetramethylsilane was used as an internal standard to set 0 ppm. Finally, compound 1 was determined as esculetin by comparing its NMR data with those reported in the reference [19]. Esculetin (1): 1H-NMR (CD3OD, 500 MHz) δ 7.76 (1H, d, J = 9.46 Hz, H-4), 6.92 (1H, s, H-5), 6.74 (1H, s, H-8), 6.16 (1H, d, J = 9.46 Hz, H-3). 13C-NMR (CD3OD, 125 MHz) δ 164.2 (C-2), 151.9 (C-9), 150.5 (C-7), 146.0 (C-4), 144.5 (C-6), 113.0 (C-5), 112.8 (C-3), 112.5 (C-10), 103.6 (C-8). Limulus Amebocyte Lysate assay was conducted to measure endotoxin levels in esculetin using QCL-1000 (BioWhittaker Bioproducts, Walkerville, MD), which was < 10 pg/mL.
To determine the effect of esculetin on DFE/DNCB-induced atopic skin inflammation, we performed repeated local exposure of the ears of mice to DFE and DNCB based on our previous research [8]. Female BALB/c mice (total 30 mice, n = 5 per group) were divided into six groups: vehicle; control, DFE/DNCB plus vehicle; AD, DFE/DNCB plus esculetin 2, 10, and 50 mg/kg, and dexamethasone (DEX) 1 mg/kg. The surfaces of both ear lobes were very gently stripped five times with a surgical tape (Nichiban, Tokyo, Japan). After stripping, total 20 μL of DFE (10 mg/mL) and 1% DNCB were applied to each ear. Vehicle (PBS) or esculetin or DEX was orally administered until the end of the 4 weeks induction (6 times per week). The control mice (n = 5) and AD mice (n = 5) were orally administered PBS alone. DFE/DNCB was alternately applied to both ears once a week for 4 weeks. Two weeks after the first induction, tail bleeding was performed to check the serum IgE level to verify the induction of atopic skin inflammation. Ear swelling was measured 24 h after DFE or DNCB application by using a dial thickness gauge (Mitutoyo, Co., Tokyo, Japan). The day before sacrifice, the mice were placed in an observation chamber with food and water for 10 min for acclimatization before the measurement of scratching. A scratching behavior was defined as hind limb scratches directed to the ears. The number of scratching events was counted for 5 min each time. On day 28, the animals were euthanized by CO2 and samples were collected for future experiments. First, blood samples were collected from the celiac artery of individual mice and centrifuged at 400g for 15 min at 4 °C for histamine assay and enzyme-linked immunosorbent assay (ELISA) including total IgE, DFE-specific IgE, and IgG2a. After blood collection, the ear tissues were removed and used for histopathological analysis and RNA extraction. 2.6. Histological observation
2.2. Animals The excised ear of mice was fixed with 10% formaldehyde and embedded in paraffin. Thin sections (5 μm) were stained with hematoxylin and eosin (H&E) and toluidine blue (TB). In skin sections
Six-week-old female BALB/c mice were purchased from SLC Inc. (Hamamatsu, Japan). The animals were housed with five mice per cage 210
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stained with H&E, infiltrated lymphocytes, thickening of the epidermis, and fibrosis in the dermis were observed by microscope as described previously [20]. Mast cell infiltration was measured in skin sections stained with TB. The number of mast cells in five sites chosen at random was counted at a magnification of ×400. The number of eosinophils in 10 high-power fields was counted in a blinded fashion at a magnification of ×400. Dermal and epidermal thicknesses were measured in H& E-stained sections viewed under a magnification of ×200. For each sample, the thickness was measured in five randomly selected fields. 2.7. Histamine assay The blood from the celiac artery of all individual mice was centrifuged at 400g for 15 min without pooling, and each serum was withdrawn to measure histamine content. Hydrochloric acid (HCl) and 60% perchloric acid were added to the serum diluted in 1/5 and subjected to centrifugation. The transferred supernatant was mixed with 5 M NaCl, 5 N NaOH and n-BuOH, and then centrifuged. The supernatant was added with HCl and n-heptane. In the last step, histamine content was measured through the o-phthaldialdehyde spectrofluorometric procedure [20]. Fluorescence intensity was measured using the fluorescence Spectrometer LS-50B (Perkin–Elmer, Norwalk, CT) with 360 nm excitation and 440 nm emission filters. 2.8. Measurement of IgE and IgG2a levels in serum The serum levels of IgE and IgG2a were measured using a specific ELISA kit (BD Biosciences, Oxford, UK) according to the standard manufacturer's instruction. Analytical sensitivity to IgE (R2 = 0.989) and IgG2a (R2 = 0.993) is than more 1.6 ng/mL and 3.1 ng/mL, respectively. The serum samples were prepared by centrifugation at 400g for 15 min without pooling and added to 96-well plates coated with single capture antibody for 24 h, respectively. After blocking of unspecific binding, samples added for 2 h. Continuously, detection was performed using biotinylated each antibodies and streptavidin conjugated HRP. The colorimetric reaction of peroxidase substrate (TMB; KPL, Gaithersburg, MD) was measured at 450 nm after the addition of 1 M sulfuric acid. Mite extract (20 μg/mL) in PBS was used as the coating solution and serum samples were aliquoted in 1/10 for the detection of DFE-specific IgE. The measurement of DFE-specific IgE was similar to that of total IgE, and DFE-specific IgE level was indicated by the optical density (O.D.) value.
Fig. 1. Effects of esculetin on ear swelling and scratching behavior of mice with DFE/DNCB-induced atopic skin inflammation. BALB/c mice (n = 5 per group) were administered vehicle (PBS) or esculetin (2, 10, or 50 mg/kg) or DEX (1 mg/kg) six times per week from day 8 after the first induction. (A) Effects of esculetin on ear swelling of mice. Weekly ear thickness was measured and recorded 24 h after DFE or DNCB application. (B) The frequency of scratching behavior was observed on the last day of induction for 5 min. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
cells/well in a 24-well plate) were pretreated with or without drugs, including esculetin (0.1, 1, or 10 μM) or DEX (10 μM) for 1 h, before stimulation with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 6 h. Total RNA was isolated using RNAiso Plus kit. qPCR was performed using SYBR Premix Ex Taq (Takarabio). The cycle number was optimized to ensure that product accumulation was in the exponential range. The conditions for PCR were similar to those as described previously. The normalization and quantification of mRNA expression were performed using the TP850 software supplied by the manufacturer. The mRNA expression levels were normalized to β-actin or GAPDH levels in each sample.
2.9. Detection of IL-6 and CCL17 in HaCaT cells IL-6 and CCL17 levels in HaCaT cells were measured using a specific ELISA kit (R&D Systems). The cells (2 × 105 cells/well in a 24-well plate) were pretreated with or without drugs, including esculetin (0.1, 1, or 10 μM) or DEX (10 μM) for 1 h, and then stimulated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 15 h. These supernatants were collected and then centrifuged at 400 g for 5 min. IL-6 and CCL17 levels were analyzed in accordance with the manufacturer's instructions. Standard curves were constructed using standard samples. The 96-well plates coated with specific each capture antibody for 24 h and samples were added for 4 h after blocking. Detection was performed with 450 nm emission filter.
2.11. Western blot HaCaT cells (1 × 106 cells/well in a 6-well plate) were pretreated with or without drugs, including esculetin (10 μM) or DEX (10 μM) for 1 h, followed by treatment with TNF-α (10 ng/mL) and IFN-γ (10 ng/ mL) for 30 min for activation of signal transducer and activator of transcription 1 (STAT1) and nuclear factor (NF)-κB (p65). After stimulation, the cells were washed twice in 2 mL of ice-cold PBS, and the lysates were collected in 100 μL of lysis buffer. The composition of the buffer has been described in previous experiments [21]. The lysates were spun in a micro-centrifuge for 30 min at 4 °C, and the supernatant
2.10. qPCR Quantitative real-time PCR (qPCR) was performed using the Thermal Cycler Dice TP850 (Takarabio, Shiga, Japan) according to the manufacturer's protocol. To detect the expression of cytokines, RNA samples were isolated from mouse ear tissues and HaCaT cells respectively. The ears of mice (total n = 60) were prepared at the end of in vivo experimental period and were 1/4 of the ear tissue of the mouse homogenized with RNAiso Plus kit (Takarabio). HaCaT cells (2 × 105 211
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Fig. 2. Representative photographs and histological aspects of DFE/DNCB-induced atopic skin inflammation. (A) Representative photographs showing skin lesions in the different groups of experimental mice (upper panel). Photomicrographs of ear sections stained with hematoxylin and eosin (H&E, middle panel) or toluidine blue (TB, lower panel). All photomicrographs are shown at the same magnification of ×400. (B) Epidermal and dermal thicknesses were analyzed in H&E-stained skin sections at ×200 magnification. (C and D) Numbers of eosinophils and mast cells were counted in H&Estained skin sections and TB-stained skin sections at ×400 magnification, respectively. The number of cells is presented as the mean of the number of cells at five random sites in each H&E and TB-stained slide. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone. Scale bars: 100 μm.
between groups were considered to be significant at a value of p < 0.05.
was collected. Equal volumes of whole-cell lysates were loaded by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membranes were stained with reversible Ponceau S to confirm equal loading of samples in the gel. The membranes were assayed using anti-NF-κB (p65), anti-IκBα, anti-lamin B1 and anti-β-actin antibodies (Santa Cruz Biotech). The phosphorylation of STAT1 was determined using anti-phospho-STAT1 (Cell Signaling, Beverly, MA). Immunodetection was performed using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ).
3. Results 3.1. Effects of esculetin on DFE/DNCB-induced atopic skin inflammation The chemical structure of esculetin and the overall experimental procedure are shown in Fig. S1A and B. In this study, we considered swelling and itching, common symptoms of AD. To determine the effects of esculetin on atopic skin inflammation, we first measured the ear thickness at 24 h after DFE/DNCB challenge. While DFE/DNCB treatment kept on increasing after 9 days of atopic skin inflammation induction, the oral administration of esculetin indicated a marked reduction in ear swelling in a dose-dependent manner (Fig. 1A). Furthermore, esculetin decreased DFE/DNCB-induced scratching
2.12. Statistical analysis Statistical analyses were performed using Prism 5 (GraphPad Software, San Diego, CA). The treatment effects were analyzed using one-way analysis of variance followed by Dunnett's test. Differences 212
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Fig. 3. Effects of esculetin on serum histamine and immunoglobulin levels in DFE/DNCB-induced atopic skin inflammation. Blood samples were collected from the celiac artery after 28 days in the different groups of experimental mice to measure histamine content and immunoglobulin levels. (A) Serum histamine levels. Each graph represents the expression patterns by concentration of (B) total IgE levels, (C) DFE-specific IgE levels, and (D) total IgG2a levels in serum. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
Fig. 4. Effects of esculetin on mRNA expression of cytokines in the ear tissue. Both ears (total n = 60) were excised and RNA was isolated. The mRNA expression of cytokines in the skin lesion was measured using qPCR. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
hyperplasia, and infiltration of inflammatory cells, compared to DFE/ DNCB-induced atopic skin inflammation (Fig. 2A). Epidermal and dermal thickness, as well as infiltration of eosinophils was significantly reduced by esculetin when compared to that observed in DFE/DNCB-
behavior (Fig. 1B). No change in body weight was observed for 4 weeks, indicating that esculetin did not cause toxicity (Fig. S1C). In histopathologic analysis, oral administration of esculetin significantly suppressed histological findings, which are epidermal 213
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Fig. 5. Effects of esculetin on gene expression and secretion of cytokines and chemokine in keratinocytes. Gene expression and secretion levels of cytokines and chemokine in TNF-α/ IFN-γ-stimulated keratinocytes were determined by qPCR and ELISA, respectively. (A) The cells were pretreated with or without drugs, including esculetin or DEX for 1 h and then stimulated with TNF-α (10 ng/ mL) and IFN-γ (10 ng/mL) for 6 h. TNFα, IL-1β, IL-6, and CCL17 mRNA expressions were determined by qPCR. (B) The cells were pretreated with esculetin or DEX for 1 h followed by stimulation with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 15 h. The protein levels of hIL-6 and hCCL17 were measured by ELISA. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
3.3. Effects of esculetin on activated keratinocytes
induced atopic skin inflammation (Fig. 2B and C). Esculetin treatment markedly the reduced accumulation of mast cells, affect the pathogenesis of AD, in the dermis of mice with AD in a dose-dependent manner (Fig. 2D).
We investigated the effects of esculetin on keratinocytes stimulated with TNF-α/IFN-γ. Preferentially, to rule out the cytotoxic effect of esculetin, cell viability was measured by the quantitative colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Treatment with esculetin as concentrations above 100 μM showed cytotoxicity; therefore, we used esculetin at concentrations < 10 μM (Fig. S1D). To determine the inhibitory effects of esculetin on gene expression and cytokine secretion in TNF-α/IFN-γ-activated keratinocytes, qPCR and ELISA were performed, respectively (Fig. 5A and B). Esculetin treatment inhibited both pro-inflammatory cytokines (TNF-α, IL-1β, IL6) and chemokine (CCL17) in TNF-α/IFN-γ-stimulated keratinocytes. To elucidate the mechanism underlying the inhibitory effects of esculetin on cytokine and chemokine expression, the activation of STAT1 and NF-κB (p65) was analyzed. Pre-treatment with esculetin (10 μM) significantly reduced TNF-α/IFN-γ-induced phosphorylation of STAT1 and nuclear translocation of NF-κB (p65) following degradation of IκBα (Fig. 6).
3.2. Effects of esculetin on the levels of histamine and immunoglobulins in serum, and pro-inflammatory cytokines in ear tissue To determine the effects of esculetin on the up-regulation of histamine and immunoglobulin levels, which are characteristic of AD, we measured serum levels. Our study showed that DFE/DNCB application increased serum histamine and immunoglobulin levels and while oral administration of esculetin suppressed the serum histamine (Fig. 3A). To distinguish the role of esculetin on Th1 and Th2 responses, serum IgE and IgG2a levels were measured by ELISA. In order to confirm that the increased serum IgE level is a response by DFE, we measured DFEspecific IgE levels. Esculetin decreased the levels of total IgE, DEFspecific IgE, and IgG2a (Fig. 3B–D). To investigate the effects of esculetin on cytokine expression in DFE/DNCB-induced atopic skin inflammation, we measured the expression of Th1 cytokines (TNF-α, IFNγ), Th2 cytokines (IL-4, IL-13, IL-31), and Th17 cytokine (IL-17A) in the ear tissue. Compared with control mice, DFE/DNCB-induced AD mice exhibited a marked increase in the RNA expression of TNF-α, IFN-γ, IL4, IL-13, IL-31, and IL-17A; however, this increase in cytokine levels was suppressed by the oral administration of esculetin (Fig. 4).
4. Discussion Fraxinus rhynchophylla is a traditional medicinal plant and widespread across the Asia, Europe, and North America [22]. Owing to 214
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activated [32]. IL-4 and IL-13, as inducer for differentiating naïve T cells into Th2, induce the isotype switching to IgE in B cell and accelerate the infiltration of inflammatory cells [8]. IgE, a hallmark of AD, recognizes allergens and sensitizes mast cells [29]. IL-31, a Th2 cytokine, has similar function to IL-4 in that it induces itching and dermatitis [33]. Meanwhile, IL-17A, mainly expressed by Th17 cells, is associated with acute AD disease and induces inflammatory responses by secreting certain cytokines and chemokines [7]. The representative Th1 cytokine IFN-γ drives the formation of IgG2a and maintains chronic AD [7,8]. The oral administration of esculetin markedly decreased IgE (total IgE and DFE-specific IgE) and IgG2a levels in serum and the expression of Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13, IL-31), and Th17 (IL17A) cytokines in the ear tissues of DFE/DNCB-induced mice. These results imply that esculetin exhibited therapeutic effects in both acute and chronic atopic skin inflammation by downregulating Th1/Th2/ Th17-immune responses. Keratinocytes are the representative cells for studying AD response. They induce T lymphocyte differentiation and produce a specific set of inflammatory cytokines and chemokines after exposure to several irritants such as TNF-α and IFN-γ [20]. Co-stimulation of TNF-α and IFN-γ has been widely used for AD-related experiments [34]. In this study, we used a human keratinocyte cell line, HaCaT, to determine the role of esculetin in TNF-α/IFN-γ-stimulated keratinocytes. As expected, treatment with esculetin suppressed the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokine (CCL17), which were increased by co-stimulation. In majority of patients with AD, CCL17 is observed, and elevated levels of CCL17 cause epidermal dysfunction [35]. Therefore, CCL17 is considered an important mediator in AD. These results suggest that esculetin exert an inhibitory effect on Th1 and Th2 responses via down-regulating the expression of inflammatory cytokines and chemokine in epidermal keratinocytes. As previous study showed that the expression of AD-related inflammatory genes are induced by the activation of STAT1 and NF-κB, which are important transcription factors in the inflammatory response of AD in HaCaT cells [36]. When the STAT1 and NF-κB pathway are activated by pro-inflammatory cytokines, STAT1 and NF-κB in keratinocytes are translocated from the cytosol into the nucleus, where they contribute to the expression of AD-related cytokines genes [37]. Therefore, the inhibition of STAT1 and NF-κB signaling pathway can alleviate AD symptoms and serve as significant targets in the development of novel therapeutics for AD [37]. Esculetin treatment prevented TNF-α/IFN-γ-induced STAT1 phosphorylation, IκBα degradation and NF-κB nuclear translocation. These results demonstrated that esculetin suppressed the activation of keratinocytes such as inflammatory cytokines and chemokine production via blockade of STAT1 and NF-κB of signaling pathway.
Fig. 6. Effects of esculetin on STAT1 and NF-κB protein levels in keratinocytes. The cells were pretreated with or without drugs, including esculetin or DEX, for 1 h, before being stimulated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 30 min. The phosphorylation of STAT1, degradation of IκBα and nuclear translocation of NF-κB were analyzed by Western blot. The data shown are a representative of three independent experiments. β-actin and lamin B1 were used as loading controls. N-NF-κB, nucleus NF-κB. DEX: dexamethasone.
various medicinal effects of F. rhynchophylla extracts; i.e., anti-asthmatic, anti-liver fibrosis, and anti-diabetes effects, there has been an increasing focus on several F. rhynchophylla extract as pharmaceutical medicines [23–25]. Esculetin isolated from F. rhynchophylla has been shown to exert therapeutic activities in the prevention of various diseases, such as cancer, nociceptive pain, and inflammation [13,15,26,27]. In particular, the anti-inflammatory effects of esculetin on asthma have been reported and it inhibits Th2 and Th17 responses and other immune responses of asthma, such as infiltration of eosinophils in bronchoalveolar lavage fluid [18]. In the present study, we demonstrated the inhibitory effects of esculetin on DFE/DNCB-induced atopic skin inflammation. BALB/c mouse model using DFE/DNCB has been used in previous studies [20]. DFE plays an important role in inflammatory reactions and is involved in the development of AD [27]. A previous study reported that repeated DFE/DNCB-application leads to common symptoms of atopic skin inflammation including swelling, pruritus, itching, dermal and epidermal thickening, and inflammatory cell (eosinophils and mast cells) infiltration [20]. Infiltration of eosinophils and mast cells into the inflammatory skin lesion is major features of AD by inducing inflammatory cytokines and chemokines [28,29]. Especially, when mast cells are sensitized by antigens cross-linking with adjacent IgE antibodies, they degranulate and secrete inflammatory mediators such as cytokines and histamine [29,30]. Histamine is found readily and at high levels in patients with AD compared to healthy controls. It is mainly derived from mast cells and plays important roles in the development of AD by exerting immunoregulatory effects and driving the development of eczematous lesions as well as itching [31]. In this study, we observed that esculetin dose-dependently reduced the symptoms of DFE/DNCBinduced atopic skin inflammation and histological changes. These results suggest that esculetin could be effective in mitigating inflammation response in AD by suppressing the infiltration of inflammatory cells. Both Th1 and Th2 cytokines are critical in the development of AD, and the balance of Th1, Th2, and Th17 cell responses in AD is mainly determined by cytokines and the signal transduction [4]. In the acute phase of AD, Th2 cytokines (IL-4, IL-13, and IL-31) are mainly expressed and in the chronic phase, Th1 cytokines (TNF-α and IFN-γ) are
5. Conclusion Esculetin showed inhibitory effects on DFE/DNCB-induced atopic skin inflammation through suppression of inflammatory cell infiltration and Th1, Th2 and Th17 cytokines expression. These results indicate that esculetin significantly suppresses both the acute and chronic atopic skin inflammation. Furthermore, esculetin blocked the activation of STAT1 and NF-κB signaling pathway, which is associated with gene expression of inflammatory mediators in keratinocytes. Based on these results, esculetin could be a critical contributor toward the treatment the skin inflammation and a potential pharmacological agent for preventing AD.
Conflict of interest The authors declare that there is no conflict of interest.
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Acknowledgements [17]
The work was supported by a National Research Foundation of Korea grant funded by the Korean Government (2014R1A5A2009242, 2016R1A2B4008513, 2015R1D1A3A01016229), KRIBB Research Initiative Program (KGM4251723), and High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
[18] [19]
[20]
Appendix A. Supplementary data
[21]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2018.04.005.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Esculetin from Fraxinus rhynchophylla attenuates atopic skin inflammation by inhibiting the expression of inflammatory cytokines
T
Na-Hee Jeonga,1, Eun-Ju Yangb,1, Meiling Jina, Jong Yeong Leea, Young-Ae Choia, Pil-Hoon Parkc, ⁎ Sang-Rae Leed, Sun-Uk Kimd, Tae-Yong Shine, Taeg Kyu Kwonf, Yong Hyun Jangg, , ⁎⁎ ⁎⁎⁎ Kyung-Sik Songb, , Sang-Hyun Kima, a
Cell & Matrix Research Institute, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea c College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea d National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea e College of Pharmacy, Woosuk University, Jeonju, Republic of Korea f Department of Immunology and School of Medicine, Keimyung University, Daegu, Republic of Korea g Department of Dermatology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Atopic dermatitis Esculetin Keratinocytes House dust mite
Atopic dermatitis (AD) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. We aimed to investigate the effects of esculetin from Fraxinus rhynchophylla on atopic skin inflammation. For induction of atopic skin inflammation, we exposed the ears of female BALB/c mice to house dust mite (Dermatophagoides farinae extract, DFE) and 2,4-dinitrochlorobenzene (DNCB) for 4 weeks. Oral administration of esculetin reduced the symptoms of DFE/DNCB-induced atopic skin inflammation, which were evaluated based on ear swelling and number of scratch bouts. The immunoglobulin (Ig) E, IgG2a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. It suppressed production of Th1, Th2 and Th17-related cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, IL-13, IL-31 and IL-17 in the ear tissue. Furthermore, we investigated the effects of esculetin on activated keratinocytes, which are representative cells used for studying the pathogenesis of acute and chronic atopic skin inflammation. As results, esculetin suppressed gene expression of Th1, Th2 and Th17 cytokines and the activation of nuclear factor-κB and signal transducer and activator of transcription 1 in TNF-α/IFN-γ-stimulated keratinocytes. Taken together, these results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin could be a potential therapeutic candidate for the treatment of AD.
1. Introduction Atopic dermatitis (AD) is a well-known allergic disorder and a common chronic inflammation disease characterized by severs, prolonged itching, lichenification, papules, excoriations and recurrent eczematous lesions [1]. AD affects millions of children and adults worldwide, which often reduces the quality of life of patients markedly. The pathogenesis of AD seems to be caused by a complex interaction between the immune system and skin barrier [2]. The pathogenesis of AD involves T helper (Th) cell immune
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responses, and Th1 and Th2 cytokines have been reported to play an important role in skin inflammation specifically AD [3]. AD is classified into two phases: acute and chronic AD. In the acute phase, Th2 and Th17 cells are mainly activated, whereas in the chronic phase, Th1 cells are mainly activated [4]. A representative Th2 cytokine interleukin (IL)-4 is an activator and driver of AD, and initiates immunoglobulin (Ig) E isotype class switching from B cells [5]. Recently, elevated levels of IL-17A produced by Th17 cells were reported in skin lesions of patients with AD, which in turn led to the release of pro-inflammatory cytokines and chemokines [6]. Then, typical Th1 cytokine interferon
Corresponding author at: Department of Dermatology, School of Medicine, Kyungpook National University, #680, Gukchaebosang-ro, Jung-gu, Daegu 41944, Republic of Korea. Corresponding author at: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Daegu 41566, Republic of Korea. Corresponding author at: Department of Pharmacology, School of Medicine, Kyungpook National University, #680, Gukchaebosang-ro, Jung-gu, Daegu 41944, Republic of Korea. E-mail addresses: [email protected] (Y.H. Jang), [email protected] (K.-S. Song), [email protected] (S.-H. Kim). 1 These authors contributed equally to this work. ⁎⁎
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https://doi.org/10.1016/j.intimp.2018.04.005 Received 16 November 2017; Received in revised form 1 March 2018; Accepted 4 April 2018 1567-5769/ © 2018 Elsevier B.V. All rights reserved.
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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 mice were provided ad libitum access to food and water, and kept on a 12 h light:dark cycle. All care and treatment of the mice were in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kyungpook National University.
(IFN)-γ contributes to the infiltration of inflammatory cells that exacerbate chronic AD and leads to the production of IgG2a [7,8]. The most commonly used therapeutic agents for AD are topical corticosteroids, immunosuppressants, anti-histamines, anti-biotics, and calcineurin inhibitors. They rapidly suppress clinical responses safely and effectively in patients with AD and are cost effective [9]. However, continuous application of corticosteroids and immunosuppressants causes serious adverse effects, such as skin atrophy or thinning, contact dermatitis, telangiectases, purpura, and striae formation [10]. Therefore, there has been an increasing focus on natural therapeutic agents as alternative medicines, because they can provide an immunomodulatory effects with fewer side effects [11]. Esculetin (also known as aesculetin, 6,7-dihydroxycoumarin or cichorigenin), a derivative of coumarin, is a phenolic compound and exists in many plants known to have medicinal effects, such as Artemisia capillaris (Compositae), Citrus limonia (Rutaceae), Ceratostigma willmottianum (Plumbaginaceae), and Fraxinus rhynchophylla (Oleaceae) [12–14]. Esculetin has been known to exhibit anti-inflammatory activity in acute lung injury, anti-nociceptive effect in inflammatory pain, anti-oxidative effect in human colon cancer, anti-cancer effect in human gastric cancer, and effectiveness against allergic asthma [13,15–18]. In this study, we investigated the pharmacological effects of esculetin isolated from F. rhynchophylla on Dermatophagoides farinae extract (DFE) and 2,4-dinitrochlorobenzene (DNCB)-induced atopic skin inflammation in a mouse model and in keratinocytes.
2.3. Chemicals DFE (Greer Laboratories, Lenoir, NC) and DNCB were used as antigen and hapten respectively, for the induction of atopic skin inflammation. All other reagents were purchased from Sigma-Aldrich unless otherwise stated. DFE was dissolved in phosphate-buffered saline (PBS) containing 0.5% Tween 20. DNCB (1%) was dissolved in an acetone/olive oil (1:3) solution. Antibiotics, and trypsin-EDTA were obtained from Invitrogen (Grand Island, NY). Recombinant tumor necrosis factor (TNF)-α and IFN-γ were purchased from R&D systems (Minneapolis, MN). 2.4. Cell culture Human HaCaT keratinocytes were maintained in Dulbecco's Modified Eagle's Medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and antibiotics (100 U/mL penicillin G, 100 μg/mL streptomycin) at 37 °C in 5% carbon dioxide (CO2). Passages 3 to 6 were used throughout the study.
2. Materials and methods 2.1. Extraction, isolation, and identification
2.5. Induction of atopic skin inflammation in mouse ear The dried stem barks (4 kg) of F. rhynchophylla purchased from the local market (Daegu, Republic of Korea), were refluxed twice with 95% ethanol (EtOH) for 2 h. The extracted solution was filtered through a filter paper (Advantec, Tokyo, Japan), and the filtrate was concentrated to dryness by a rotary evaporator (EYELA, Tokyo, Japan) under reduced pressure below 40 °C. The EtOH extract (510 g) was suspended in distilled water, and successively partitioned with dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). The EtOAcsoluble fraction (45 g) was applied to a silica gel (70–230 mesh, Merck, Darmstadt, Germany) column [Ø 5.5 × 83 cm, chloroform (CHCl3): methanol (MeOH) = 20:1–11] to obtain nine fractions (Fr. 1-9). Fr. 5 was rechromatographed by using silica gel (70–230 mesh, Ø 2.5 × 83 cm, CH2Cl2: MeOH = 20:1-1:1) to yield six subfractions (Fr. 51 to 5-6). Compound 1 (20 mg) was isolated as an amorphous yellow powder via silica gel column chromatography (230–400 mesh, Ø 1 × 60 cm, EtOAc:MeOH:H2O = 100:1:1% - 10:1:1%) from Fr. 5-4. To identify the isolated compound 1, nuclear magnetic resonance (NMR; Bruker Avance Digital 500 NMR spectrometer, Karlsruhe, Germany) analysis was performed. Compound 1 was dissolved in deuterated methanol (CD3OD, Sigma-Aldrich, St. Louis, MO) before NMR analysis, and tetramethylsilane was used as an internal standard to set 0 ppm. Finally, compound 1 was determined as esculetin by comparing its NMR data with those reported in the reference [19]. Esculetin (1): 1H-NMR (CD3OD, 500 MHz) δ 7.76 (1H, d, J = 9.46 Hz, H-4), 6.92 (1H, s, H-5), 6.74 (1H, s, H-8), 6.16 (1H, d, J = 9.46 Hz, H-3). 13C-NMR (CD3OD, 125 MHz) δ 164.2 (C-2), 151.9 (C-9), 150.5 (C-7), 146.0 (C-4), 144.5 (C-6), 113.0 (C-5), 112.8 (C-3), 112.5 (C-10), 103.6 (C-8). Limulus Amebocyte Lysate assay was conducted to measure endotoxin levels in esculetin using QCL-1000 (BioWhittaker Bioproducts, Walkerville, MD), which was < 10 pg/mL.
To determine the effect of esculetin on DFE/DNCB-induced atopic skin inflammation, we performed repeated local exposure of the ears of mice to DFE and DNCB based on our previous research [8]. Female BALB/c mice (total 30 mice, n = 5 per group) were divided into six groups: vehicle; control, DFE/DNCB plus vehicle; AD, DFE/DNCB plus esculetin 2, 10, and 50 mg/kg, and dexamethasone (DEX) 1 mg/kg. The surfaces of both ear lobes were very gently stripped five times with a surgical tape (Nichiban, Tokyo, Japan). After stripping, total 20 μL of DFE (10 mg/mL) and 1% DNCB were applied to each ear. Vehicle (PBS) or esculetin or DEX was orally administered until the end of the 4 weeks induction (6 times per week). The control mice (n = 5) and AD mice (n = 5) were orally administered PBS alone. DFE/DNCB was alternately applied to both ears once a week for 4 weeks. Two weeks after the first induction, tail bleeding was performed to check the serum IgE level to verify the induction of atopic skin inflammation. Ear swelling was measured 24 h after DFE or DNCB application by using a dial thickness gauge (Mitutoyo, Co., Tokyo, Japan). The day before sacrifice, the mice were placed in an observation chamber with food and water for 10 min for acclimatization before the measurement of scratching. A scratching behavior was defined as hind limb scratches directed to the ears. The number of scratching events was counted for 5 min each time. On day 28, the animals were euthanized by CO2 and samples were collected for future experiments. First, blood samples were collected from the celiac artery of individual mice and centrifuged at 400g for 15 min at 4 °C for histamine assay and enzyme-linked immunosorbent assay (ELISA) including total IgE, DFE-specific IgE, and IgG2a. After blood collection, the ear tissues were removed and used for histopathological analysis and RNA extraction. 2.6. Histological observation
2.2. Animals The excised ear of mice was fixed with 10% formaldehyde and embedded in paraffin. Thin sections (5 μm) were stained with hematoxylin and eosin (H&E) and toluidine blue (TB). In skin sections
Six-week-old female BALB/c mice were purchased from SLC Inc. (Hamamatsu, Japan). The animals were housed with five mice per cage 210
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stained with H&E, infiltrated lymphocytes, thickening of the epidermis, and fibrosis in the dermis were observed by microscope as described previously [20]. Mast cell infiltration was measured in skin sections stained with TB. The number of mast cells in five sites chosen at random was counted at a magnification of ×400. The number of eosinophils in 10 high-power fields was counted in a blinded fashion at a magnification of ×400. Dermal and epidermal thicknesses were measured in H& E-stained sections viewed under a magnification of ×200. For each sample, the thickness was measured in five randomly selected fields. 2.7. Histamine assay The blood from the celiac artery of all individual mice was centrifuged at 400g for 15 min without pooling, and each serum was withdrawn to measure histamine content. Hydrochloric acid (HCl) and 60% perchloric acid were added to the serum diluted in 1/5 and subjected to centrifugation. The transferred supernatant was mixed with 5 M NaCl, 5 N NaOH and n-BuOH, and then centrifuged. The supernatant was added with HCl and n-heptane. In the last step, histamine content was measured through the o-phthaldialdehyde spectrofluorometric procedure [20]. Fluorescence intensity was measured using the fluorescence Spectrometer LS-50B (Perkin–Elmer, Norwalk, CT) with 360 nm excitation and 440 nm emission filters. 2.8. Measurement of IgE and IgG2a levels in serum The serum levels of IgE and IgG2a were measured using a specific ELISA kit (BD Biosciences, Oxford, UK) according to the standard manufacturer's instruction. Analytical sensitivity to IgE (R2 = 0.989) and IgG2a (R2 = 0.993) is than more 1.6 ng/mL and 3.1 ng/mL, respectively. The serum samples were prepared by centrifugation at 400g for 15 min without pooling and added to 96-well plates coated with single capture antibody for 24 h, respectively. After blocking of unspecific binding, samples added for 2 h. Continuously, detection was performed using biotinylated each antibodies and streptavidin conjugated HRP. The colorimetric reaction of peroxidase substrate (TMB; KPL, Gaithersburg, MD) was measured at 450 nm after the addition of 1 M sulfuric acid. Mite extract (20 μg/mL) in PBS was used as the coating solution and serum samples were aliquoted in 1/10 for the detection of DFE-specific IgE. The measurement of DFE-specific IgE was similar to that of total IgE, and DFE-specific IgE level was indicated by the optical density (O.D.) value.
Fig. 1. Effects of esculetin on ear swelling and scratching behavior of mice with DFE/DNCB-induced atopic skin inflammation. BALB/c mice (n = 5 per group) were administered vehicle (PBS) or esculetin (2, 10, or 50 mg/kg) or DEX (1 mg/kg) six times per week from day 8 after the first induction. (A) Effects of esculetin on ear swelling of mice. Weekly ear thickness was measured and recorded 24 h after DFE or DNCB application. (B) The frequency of scratching behavior was observed on the last day of induction for 5 min. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
cells/well in a 24-well plate) were pretreated with or without drugs, including esculetin (0.1, 1, or 10 μM) or DEX (10 μM) for 1 h, before stimulation with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 6 h. Total RNA was isolated using RNAiso Plus kit. qPCR was performed using SYBR Premix Ex Taq (Takarabio). The cycle number was optimized to ensure that product accumulation was in the exponential range. The conditions for PCR were similar to those as described previously. The normalization and quantification of mRNA expression were performed using the TP850 software supplied by the manufacturer. The mRNA expression levels were normalized to β-actin or GAPDH levels in each sample.
2.9. Detection of IL-6 and CCL17 in HaCaT cells IL-6 and CCL17 levels in HaCaT cells were measured using a specific ELISA kit (R&D Systems). The cells (2 × 105 cells/well in a 24-well plate) were pretreated with or without drugs, including esculetin (0.1, 1, or 10 μM) or DEX (10 μM) for 1 h, and then stimulated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 15 h. These supernatants were collected and then centrifuged at 400 g for 5 min. IL-6 and CCL17 levels were analyzed in accordance with the manufacturer's instructions. Standard curves were constructed using standard samples. The 96-well plates coated with specific each capture antibody for 24 h and samples were added for 4 h after blocking. Detection was performed with 450 nm emission filter.
2.11. Western blot HaCaT cells (1 × 106 cells/well in a 6-well plate) were pretreated with or without drugs, including esculetin (10 μM) or DEX (10 μM) for 1 h, followed by treatment with TNF-α (10 ng/mL) and IFN-γ (10 ng/ mL) for 30 min for activation of signal transducer and activator of transcription 1 (STAT1) and nuclear factor (NF)-κB (p65). After stimulation, the cells were washed twice in 2 mL of ice-cold PBS, and the lysates were collected in 100 μL of lysis buffer. The composition of the buffer has been described in previous experiments [21]. The lysates were spun in a micro-centrifuge for 30 min at 4 °C, and the supernatant
2.10. qPCR Quantitative real-time PCR (qPCR) was performed using the Thermal Cycler Dice TP850 (Takarabio, Shiga, Japan) according to the manufacturer's protocol. To detect the expression of cytokines, RNA samples were isolated from mouse ear tissues and HaCaT cells respectively. The ears of mice (total n = 60) were prepared at the end of in vivo experimental period and were 1/4 of the ear tissue of the mouse homogenized with RNAiso Plus kit (Takarabio). HaCaT cells (2 × 105 211
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Fig. 2. Representative photographs and histological aspects of DFE/DNCB-induced atopic skin inflammation. (A) Representative photographs showing skin lesions in the different groups of experimental mice (upper panel). Photomicrographs of ear sections stained with hematoxylin and eosin (H&E, middle panel) or toluidine blue (TB, lower panel). All photomicrographs are shown at the same magnification of ×400. (B) Epidermal and dermal thicknesses were analyzed in H&E-stained skin sections at ×200 magnification. (C and D) Numbers of eosinophils and mast cells were counted in H&Estained skin sections and TB-stained skin sections at ×400 magnification, respectively. The number of cells is presented as the mean of the number of cells at five random sites in each H&E and TB-stained slide. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone. Scale bars: 100 μm.
between groups were considered to be significant at a value of p < 0.05.
was collected. Equal volumes of whole-cell lysates were loaded by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membranes were stained with reversible Ponceau S to confirm equal loading of samples in the gel. The membranes were assayed using anti-NF-κB (p65), anti-IκBα, anti-lamin B1 and anti-β-actin antibodies (Santa Cruz Biotech). The phosphorylation of STAT1 was determined using anti-phospho-STAT1 (Cell Signaling, Beverly, MA). Immunodetection was performed using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ).
3. Results 3.1. Effects of esculetin on DFE/DNCB-induced atopic skin inflammation The chemical structure of esculetin and the overall experimental procedure are shown in Fig. S1A and B. In this study, we considered swelling and itching, common symptoms of AD. To determine the effects of esculetin on atopic skin inflammation, we first measured the ear thickness at 24 h after DFE/DNCB challenge. While DFE/DNCB treatment kept on increasing after 9 days of atopic skin inflammation induction, the oral administration of esculetin indicated a marked reduction in ear swelling in a dose-dependent manner (Fig. 1A). Furthermore, esculetin decreased DFE/DNCB-induced scratching
2.12. Statistical analysis Statistical analyses were performed using Prism 5 (GraphPad Software, San Diego, CA). The treatment effects were analyzed using one-way analysis of variance followed by Dunnett's test. Differences 212
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Fig. 3. Effects of esculetin on serum histamine and immunoglobulin levels in DFE/DNCB-induced atopic skin inflammation. Blood samples were collected from the celiac artery after 28 days in the different groups of experimental mice to measure histamine content and immunoglobulin levels. (A) Serum histamine levels. Each graph represents the expression patterns by concentration of (B) total IgE levels, (C) DFE-specific IgE levels, and (D) total IgG2a levels in serum. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
Fig. 4. Effects of esculetin on mRNA expression of cytokines in the ear tissue. Both ears (total n = 60) were excised and RNA was isolated. The mRNA expression of cytokines in the skin lesion was measured using qPCR. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
hyperplasia, and infiltration of inflammatory cells, compared to DFE/ DNCB-induced atopic skin inflammation (Fig. 2A). Epidermal and dermal thickness, as well as infiltration of eosinophils was significantly reduced by esculetin when compared to that observed in DFE/DNCB-
behavior (Fig. 1B). No change in body weight was observed for 4 weeks, indicating that esculetin did not cause toxicity (Fig. S1C). In histopathologic analysis, oral administration of esculetin significantly suppressed histological findings, which are epidermal 213
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Fig. 5. Effects of esculetin on gene expression and secretion of cytokines and chemokine in keratinocytes. Gene expression and secretion levels of cytokines and chemokine in TNF-α/ IFN-γ-stimulated keratinocytes were determined by qPCR and ELISA, respectively. (A) The cells were pretreated with or without drugs, including esculetin or DEX for 1 h and then stimulated with TNF-α (10 ng/ mL) and IFN-γ (10 ng/mL) for 6 h. TNFα, IL-1β, IL-6, and CCL17 mRNA expressions were determined by qPCR. (B) The cells were pretreated with esculetin or DEX for 1 h followed by stimulation with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 15 h. The protein levels of hIL-6 and hCCL17 were measured by ELISA. Data are presented as mean ± SD (n = 5). *Significantly lower than those of AD mice at p < 0.05. DEX: dexamethasone.
3.3. Effects of esculetin on activated keratinocytes
induced atopic skin inflammation (Fig. 2B and C). Esculetin treatment markedly the reduced accumulation of mast cells, affect the pathogenesis of AD, in the dermis of mice with AD in a dose-dependent manner (Fig. 2D).
We investigated the effects of esculetin on keratinocytes stimulated with TNF-α/IFN-γ. Preferentially, to rule out the cytotoxic effect of esculetin, cell viability was measured by the quantitative colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Treatment with esculetin as concentrations above 100 μM showed cytotoxicity; therefore, we used esculetin at concentrations < 10 μM (Fig. S1D). To determine the inhibitory effects of esculetin on gene expression and cytokine secretion in TNF-α/IFN-γ-activated keratinocytes, qPCR and ELISA were performed, respectively (Fig. 5A and B). Esculetin treatment inhibited both pro-inflammatory cytokines (TNF-α, IL-1β, IL6) and chemokine (CCL17) in TNF-α/IFN-γ-stimulated keratinocytes. To elucidate the mechanism underlying the inhibitory effects of esculetin on cytokine and chemokine expression, the activation of STAT1 and NF-κB (p65) was analyzed. Pre-treatment with esculetin (10 μM) significantly reduced TNF-α/IFN-γ-induced phosphorylation of STAT1 and nuclear translocation of NF-κB (p65) following degradation of IκBα (Fig. 6).
3.2. Effects of esculetin on the levels of histamine and immunoglobulins in serum, and pro-inflammatory cytokines in ear tissue To determine the effects of esculetin on the up-regulation of histamine and immunoglobulin levels, which are characteristic of AD, we measured serum levels. Our study showed that DFE/DNCB application increased serum histamine and immunoglobulin levels and while oral administration of esculetin suppressed the serum histamine (Fig. 3A). To distinguish the role of esculetin on Th1 and Th2 responses, serum IgE and IgG2a levels were measured by ELISA. In order to confirm that the increased serum IgE level is a response by DFE, we measured DFEspecific IgE levels. Esculetin decreased the levels of total IgE, DEFspecific IgE, and IgG2a (Fig. 3B–D). To investigate the effects of esculetin on cytokine expression in DFE/DNCB-induced atopic skin inflammation, we measured the expression of Th1 cytokines (TNF-α, IFNγ), Th2 cytokines (IL-4, IL-13, IL-31), and Th17 cytokine (IL-17A) in the ear tissue. Compared with control mice, DFE/DNCB-induced AD mice exhibited a marked increase in the RNA expression of TNF-α, IFN-γ, IL4, IL-13, IL-31, and IL-17A; however, this increase in cytokine levels was suppressed by the oral administration of esculetin (Fig. 4).
4. Discussion Fraxinus rhynchophylla is a traditional medicinal plant and widespread across the Asia, Europe, and North America [22]. Owing to 214
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activated [32]. IL-4 and IL-13, as inducer for differentiating naïve T cells into Th2, induce the isotype switching to IgE in B cell and accelerate the infiltration of inflammatory cells [8]. IgE, a hallmark of AD, recognizes allergens and sensitizes mast cells [29]. IL-31, a Th2 cytokine, has similar function to IL-4 in that it induces itching and dermatitis [33]. Meanwhile, IL-17A, mainly expressed by Th17 cells, is associated with acute AD disease and induces inflammatory responses by secreting certain cytokines and chemokines [7]. The representative Th1 cytokine IFN-γ drives the formation of IgG2a and maintains chronic AD [7,8]. The oral administration of esculetin markedly decreased IgE (total IgE and DFE-specific IgE) and IgG2a levels in serum and the expression of Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13, IL-31), and Th17 (IL17A) cytokines in the ear tissues of DFE/DNCB-induced mice. These results imply that esculetin exhibited therapeutic effects in both acute and chronic atopic skin inflammation by downregulating Th1/Th2/ Th17-immune responses. Keratinocytes are the representative cells for studying AD response. They induce T lymphocyte differentiation and produce a specific set of inflammatory cytokines and chemokines after exposure to several irritants such as TNF-α and IFN-γ [20]. Co-stimulation of TNF-α and IFN-γ has been widely used for AD-related experiments [34]. In this study, we used a human keratinocyte cell line, HaCaT, to determine the role of esculetin in TNF-α/IFN-γ-stimulated keratinocytes. As expected, treatment with esculetin suppressed the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokine (CCL17), which were increased by co-stimulation. In majority of patients with AD, CCL17 is observed, and elevated levels of CCL17 cause epidermal dysfunction [35]. Therefore, CCL17 is considered an important mediator in AD. These results suggest that esculetin exert an inhibitory effect on Th1 and Th2 responses via down-regulating the expression of inflammatory cytokines and chemokine in epidermal keratinocytes. As previous study showed that the expression of AD-related inflammatory genes are induced by the activation of STAT1 and NF-κB, which are important transcription factors in the inflammatory response of AD in HaCaT cells [36]. When the STAT1 and NF-κB pathway are activated by pro-inflammatory cytokines, STAT1 and NF-κB in keratinocytes are translocated from the cytosol into the nucleus, where they contribute to the expression of AD-related cytokines genes [37]. Therefore, the inhibition of STAT1 and NF-κB signaling pathway can alleviate AD symptoms and serve as significant targets in the development of novel therapeutics for AD [37]. Esculetin treatment prevented TNF-α/IFN-γ-induced STAT1 phosphorylation, IκBα degradation and NF-κB nuclear translocation. These results demonstrated that esculetin suppressed the activation of keratinocytes such as inflammatory cytokines and chemokine production via blockade of STAT1 and NF-κB of signaling pathway.
Fig. 6. Effects of esculetin on STAT1 and NF-κB protein levels in keratinocytes. The cells were pretreated with or without drugs, including esculetin or DEX, for 1 h, before being stimulated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 30 min. The phosphorylation of STAT1, degradation of IκBα and nuclear translocation of NF-κB were analyzed by Western blot. The data shown are a representative of three independent experiments. β-actin and lamin B1 were used as loading controls. N-NF-κB, nucleus NF-κB. DEX: dexamethasone.
various medicinal effects of F. rhynchophylla extracts; i.e., anti-asthmatic, anti-liver fibrosis, and anti-diabetes effects, there has been an increasing focus on several F. rhynchophylla extract as pharmaceutical medicines [23–25]. Esculetin isolated from F. rhynchophylla has been shown to exert therapeutic activities in the prevention of various diseases, such as cancer, nociceptive pain, and inflammation [13,15,26,27]. In particular, the anti-inflammatory effects of esculetin on asthma have been reported and it inhibits Th2 and Th17 responses and other immune responses of asthma, such as infiltration of eosinophils in bronchoalveolar lavage fluid [18]. In the present study, we demonstrated the inhibitory effects of esculetin on DFE/DNCB-induced atopic skin inflammation. BALB/c mouse model using DFE/DNCB has been used in previous studies [20]. DFE plays an important role in inflammatory reactions and is involved in the development of AD [27]. A previous study reported that repeated DFE/DNCB-application leads to common symptoms of atopic skin inflammation including swelling, pruritus, itching, dermal and epidermal thickening, and inflammatory cell (eosinophils and mast cells) infiltration [20]. Infiltration of eosinophils and mast cells into the inflammatory skin lesion is major features of AD by inducing inflammatory cytokines and chemokines [28,29]. Especially, when mast cells are sensitized by antigens cross-linking with adjacent IgE antibodies, they degranulate and secrete inflammatory mediators such as cytokines and histamine [29,30]. Histamine is found readily and at high levels in patients with AD compared to healthy controls. It is mainly derived from mast cells and plays important roles in the development of AD by exerting immunoregulatory effects and driving the development of eczematous lesions as well as itching [31]. In this study, we observed that esculetin dose-dependently reduced the symptoms of DFE/DNCBinduced atopic skin inflammation and histological changes. These results suggest that esculetin could be effective in mitigating inflammation response in AD by suppressing the infiltration of inflammatory cells. Both Th1 and Th2 cytokines are critical in the development of AD, and the balance of Th1, Th2, and Th17 cell responses in AD is mainly determined by cytokines and the signal transduction [4]. In the acute phase of AD, Th2 cytokines (IL-4, IL-13, and IL-31) are mainly expressed and in the chronic phase, Th1 cytokines (TNF-α and IFN-γ) are
5. Conclusion Esculetin showed inhibitory effects on DFE/DNCB-induced atopic skin inflammation through suppression of inflammatory cell infiltration and Th1, Th2 and Th17 cytokines expression. These results indicate that esculetin significantly suppresses both the acute and chronic atopic skin inflammation. Furthermore, esculetin blocked the activation of STAT1 and NF-κB signaling pathway, which is associated with gene expression of inflammatory mediators in keratinocytes. Based on these results, esculetin could be a critical contributor toward the treatment the skin inflammation and a potential pharmacological agent for preventing AD.
Conflict of interest The authors declare that there is no conflict of interest.
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Acknowledgements [17]
The work was supported by a National Research Foundation of Korea grant funded by the Korean Government (2014R1A5A2009242, 2016R1A2B4008513, 2015R1D1A3A01016229), KRIBB Research Initiative Program (KGM4251723), and High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
[18] [19]
[20]
Appendix A. Supplementary data
[21]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2018.04.005.
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