Inhibitory effects of atractylone on mast cell-mediated allergic reactions

Chemico-Biological Interactions 258 (2016) 59e68

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Inhibitory effects of atractylone on mast cell-mediated allergic reactions Na-Ra Han a, 1, Phil-Dong Moon a, 1, Sun-Young Nam a, Ka-Jung Ryu a, Myoung-Schook Yoou a, Jung-hye Choi b, Sung-Yeoun Hwang c, Hyung-Min Kim a, **, Hyun-Ja Jeong d, * a

Department of Pharmacology, College of Korean Medicine, Kyung Hee University, Seoul, 02447, Republic of Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, 02447, Republic of Korea Korea Bio Medical Science Institute, Gangnam-gu, Seoul, 06106, Republic of Korea d Department of Food Technology and Inflammatory Disease Research Center, Hoseo University, 20 Hoseo-ro, 79 Beon-gil, Baebang-eup, Asan, Chungnam, 31499, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 April 2016 Received in revised form 10 August 2016 Accepted 18 August 2016 Available online 20 August 2016

This study investigated a salutary effect of atractylone (Atr) which is an active constituent of PyeongweeSan (KMP6) on mast cell-mediated allergic reactions. Our previous report indicated that KMP6 regulated allergic reactions. Thus, this study sought to determine the potential of Atr in vitro models, compound 48/ 80-stimulated rat peritoneal mast cells (RPMCs), phorbol 12-myristate 13-acetate (PMA) plus A23187stimulated human mast cell line (HMC-1) cells, and stem cell factor (SCF)-stimulated RPMCs as well as in vivo models, IgE-mediated passive cutaneous anaphylaxis (PCA), compound 48/80-induced systemic anaphylaxis, and compound 48/80-induced ear swelling. The results showed that Atr inhibited compound 48/80-induced RPMCs degranulation, intracellular calcium level, tryptase release, and histamine release. Atr inhibited the up-regulation of p56lck tyrosine kinase activity by compound 48/80. And Atr reduced tryptase and histamine releases from PMA plus A23187-stimulated HMC-1 cells. In addition, Atr decreased histidine decarboxylase activity and expression in the activated HMC-1 cells. Atr inhibited SCF-induced morphological alteration and filamentous actin formation in RPMCs. Atr improved IgEinduced PCA reaction by decreasing the levels of histamine, IgE, interleukin (IL)-4, IL-5, IL-6, vascular endothelial growth factor, and IL-13 in the serum of PCA-induced mice. Furthermore, Atr mitigated compound 48/80-induced systemic anaphylaxis and ear swelling. Taken together, these results of this study indicate that Atr regulates the degranulation of mast cell, proving its potential in the treatment of mast cell-mediated allergic reactions. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Atractylone Mast cell Degranulation Histamine Anaphylaxis Cytokine

1. Introduction Allergic reactions can be common and mild but a lifethreatening and serious illness. Allergic reactions have developed from complex interactions, such as dietary factors or environmental factors. Disordered eating habits can influence sensitization to allergens or increase the risk of developing allergic symptoms

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H.-M. (H.-J. Jeong). 1 These authors contributed equally to this work.

Kim),

http://dx.doi.org/10.1016/j.cbi.2016.08.015 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.

[email protected]

[1]. Seriously, dyspepsia or Helicobacter pylori infection is associated with skin diseases [2,3]. Dyspepsia was manifested in patients with atopic diseases or allergic rhinitis [4]. Mast cells have an inextricable connection with the pathology of allergic disorders including fatal anaphylaxis [5]. Upon exposure to allergens, IgE-bound Fc3RI on mast cells becomes crosslinked and leads to mast cell activation via activation of tyrosine kinases [6]. The activated mast cells release tryptase or histamine as well as various inflammatory cytokines, such as interleukin (IL)-1b, IL-4, IL5, IL-6, IL-13, vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF)-a [7]. Histamine is a primary mediator of anaphylactic reactions and triggers cascade pathways of inflammatory process [8]. And it was synthesized by histidine decarboxylase (HDC).

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Stem cell factor (SCF) is a main chemotactic factor for mast cells. SCF promotes proliferation, survival, and maturation of mast cells [9]. Also, SCF exacerbates chronic severe allergic responses via the mast cell degranulation and cytokine production [10,11]. SCF results in a change in filamentous actin (F-actin) distribution around the cell rim [12]. F-actin is involved in mast cell degranulation and migration [13]. Our previous report showed that Pyeongwee-San (KMP6) which has been widely used to treat digestive disorders in Korean medicine had anti-allergic effect [14]. Atractylone (Atr, Fig 1A) is a main sesquiterpenic constituent of Atractylodes japonica which is a main component of KMP6 [15]. Atractylodes japonica was reported to have anti-inflammatory actions [16]. Atr also was reported to have anti-inflammatory effect [17] and anti-hepatotoxic effect [18]. However, the effect of Atr in mast cell-mediated allergic reactions has not been identified. In this study, we investigated the regulatory effect and its mechanism of Atr in the mast cell-mediated allergic reaction using in vitro models, compound 48/80 (an inducer of mast cell degranulation, Fig 1B)-stimulated rat peritoneal mast cells (RPMCs), SCF-stimulated RPMCs, and phorbol 12myristate 13-acetate (PMA) plus A23187 (calcium ionophore)stimulated human mast cell line (HMC-1) cells as well as in vivo models, IgE-mediated passive cutaneous anaphylaxis (PCA), compound 48/80-induced systemic anaphylaxis, and compound 48/80induced ear swelling.

(Eumsung, Republic of Korea). The animals were acclimated at 20e23  C with 50e60% humidity (12 h light-dark cycles). All experimental procedures involving animals followed the ethical regulations of the animal care committee of Kyung Hee University (No. KHUASP(SE)-15-118).

2.3. Preparation of Atr and KMP6 Atr was dissolved in 10% dimethyl sulfoxide. A dose of Atr was determined on the basis of a previous report [15]. KMP6 was obtained from Korea Bio Medical Science Institute (Seoul, Republic of Korea). A prescription of KMP6 consists of Atractylodes japonica Koidzumi (13.3 g), Magnolia officinale Rehder et Wils (10 g), Citrus sunki Hort. ex Tanaka (10 g), Zingiber officinale Roscoe (3.3 g), Glycyrrhiza uralensis Fisch (3.3 g), and Zizyphus jujuba var. inermis (Bunge) Rehder (6.7 g). An extract of KMP6 was prepared by decocting with distilled water (DW) for approximately 3 h according to previous reports [19,20]. The decocted extract has been filtered and lyophilized. The yield of KMP6 was about 21% (w/w). The KMP6 powder was dissolved with DW and filtered through a 0.22 mm syringe filter. A dose (0.1 mg/kg) of KMP6 was determined according to a previous report [14].

2.4. Mast cell culture 2. Materials and methods 2.1. Materials Atr was purchased from ChemFaces Biochemical Co., Ltd. (Hubei, China); Isocove's Modified Dulbecco's Medium (IMDM) and fetal bovine serum (FBS) from Gibco BRL (Grand Island, NY, USA); compound 48/80, percoll®, ketotifen, 1,2-bis(2-aminophenooxy) ethane-N,N,N0 ,N'- tertraacetic acid-AM (Bapta-AM), Fura-2/AM, damnacanthal (Dam), PMA, A23187 (Calcimycin; C29H37N3O6), dexamethasone (DEX), anti-dinitrophenyl (DNP)-IgE, DNP-human serum albumin (HSA), and evans blue from Sigma Chemical Co., (St. Louis, MO, USA); HDC and GAPDH antibodies from Santa Cruz Biotechnology (Dallas, Texas, USA); IgE, IL-4, IL-6, and TNF-a antibodies from BD Pharmingen (Torreyana Road, San Diego, CA, USA); IL-1b, IL-5, VEGF, and IL-13 antibodies from R&D Systems (Minneapolis, MN, USA). 2.2. Animals Male Sprague Dawley rats (7 weeks old) and ICR mice (4 weeks old) were obtained from the Dae-Han Experimental Animal Center

Rats were injected with saline buffer (30 mL) including FBS and heparin into the peritoneal cavity. The abdomen was gently massaged for about 3 min. The peritoneal cavity was opened and the fluid was aspirated using a Pasteur pipette. RPMCs were purified by a Percoll density gradient centrifugation as previously described [21]. HMC-1 cells were cultured in IMDM with FBS (10%), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37  C in 5% CO2 with 95% humidity.

2.5. Fluorescent measurements of intracellular calcium levels To evaluate the intracellular calcium level, purified RPMCs or HMC-1 cells suspensions were pretreated with Fura-2/AM for 30 min and then were harvested. After washing twice with medium containing extracellular calcium chelator EGTA (0.5 mM), the cell suspension (1  105 cells) was placed into a 96-well plate and pretreated with Atr, KMP6, ketotifen, or Bapta-AM for 20 min. RPMCs were stimulated with compound 48/80 (6 mg/mL) and HMC1 cells were stimulated with PMA plus A23187 (PMACI) for 5 min. The intracellular calcium levels were determined at 440 nm (excitation 360 nm) in a spectrofluorometer.

Fig. 1. Structure of (A) Atr and (B) compound 48/80.

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2.6. Tryptase assay

2.12. F-actin formation in RPMCs

Purified RPMCs (2  105 cells) were treated with Atr, KMP6, or ketotifen for 40 min and then stimulated for 15 min with compound 48/80 (6 mg/mL). HMC-1 cells (1  106 cells) were treated with Atr, KMP6, or ketotifen for 2 h and then activated with PMACI for 6 h. Tryptase level from each culture supernatant was determined with a mast cell degranulation assay kit (Millipore Co., Billerica, MA, USA).

RPMCs were treated with Atr, KMP6, or DEX for 1 h and stimulated with SCF for 1 h. RPMCs were fixed with 3% paraformaldehyde/phosphateebuffered saline (PBS) for 1 h, permeabilized with 1% Triton X-100/PBS for 15 min, and then stained for 30 min with F-actin specific probe, 1 U/mL NBDphallacidin. Each specimen was observed with a confocal laserscanning microscope (excitation at 488 nm).

2.7. Histamine assay

2.13. PCA reaction

Histamine levels from RPMCs, HMC-1 cells, or serum of PCAinduced mice were determined using o-phthalaldehyde spectrofluorometric analysis [22]. The fluorescent intensity was determined at 440 nm (excitation at 360 nm) in a spectrofluorometer. 2.8. RPMCs morphology Purified RPMCs suspensions were treated with Atr, KMP6, or ketotifen for 40 min and stimulated with compound 48/80 (6 mg/ mL). The morphological changes were observed using an inverted microscope. Five sections were randomly selected and the number of degranulated RPMCs was counted following report of Penissi et al. [23]. Basal RPMCs were circular in shape and released no granules. The degranulated cells showed swelling, disrupted boundaries, and multiple granules compared to the basal RPMCs. To measure the number of cells having SCF-altered morphological features, RPMCs were treated with Atr, KMP6, or DEX for 1 h and then stimulated with SCF (50 ng/mL) for 4 days. The morphology was observed under a microscope at 400  magnification. lck

2.9. p56

tyrosine kinase assay

Purified RPMCs (3  106 cells) were treated with Atr, KMP6, or Dam for 40 min and then stimulated with compound 48/80 (6 mg/ mL) for 20 min. Activity of p56lck tyrosine kinase was examined according to the manufacturer's specification of p56lck kinase assay kit (CycLex Co., Ltd., Nagano, Japan). 2.10. HDC assay HMC-1 cells (3  106 cells) were treated with Atr, KMP6, or DEX for 2 h and then stimulated with PMACI for 4 h. HMC-1 cells were lysed in potassium phosphate buffer (HDC reaction buffer, 0.1 M, pH 6.8) containing dithiothreitol (0.2 mM), pyridoxal (0.01 mM)phosphate(5%), polyethylene glycol (Mr ¼ 300, 1%), and phenylmethylsulphonyl fluoride (100 mg/mL) according to a previous report [24]. After a centrifugation, the supernatant was incubated with HDC reaction buffer containing 0.25 mM L-histidine for 2 h at 37  C. HDC activity was represented as the amount of histamine which is measured by histamine assay as described above. 2.11. Western blot analysis HMC-1 cells (3  106 cells) were treated with Atr, KMP6, or DEX for 2 h and then activated with PMACI for 6 h. The harvested HMC1 cells were lysed and resolved by 10% SDS-PAGE. After electrophoresis, the protein was transferred to nitrocellulose membranes and the membranes were blocked and incubated with anti-HDC antibody. Blots were developed by peroxidase-conjugated secondary antibodies. The protein bands were detected with an enhanced chemiluminesence assay (Amersham Co., Newark, NJ, USA).

PCA reaction was performed according to a previous report [25]. 2.14. Enzyme-linked immunosorbent assay (ELISA) IgE, IL-1b, IL-4, IL-5, IL-6, VEGF, IL-13, and TNF-a levels were determined in the serum obtained after PCA reaction with ELISA method according to the manufacturer's specifications (R & D system and BD Pharmingen). 2.15. Compound 48/80-induced systemic anaphylactic reaction Atr, KMP6, or ketotifen was orally administered 1 h before the injection of compound 48/80 (8 mg/kg) according to Kim et al. [25]. Control group was administered with saline. The mortality was measured on the basis of control mice that died about 13 min after the compound 48/80-injection. 2.16. Compound 48/80-induced ear swelling response Atr, KMP6, or ketotifen was orally administered to mice. After 1 h, compound 48/80 was injected intradermally (100 mg/site) into the dorsal side of each ear. Ear swelling response was determined 40 min after compound 48/80 or saline injection according to Kim et al. [25]. Ear thickness was determined with a digimatic micrometer (Mitutoyo, Japan). 2.17. Statistical analysis All results were analyzed using one-way ANOVA. The in vitro results are presented as mean ± standard error of the mean (SEM) from at least three independent experiments with duplicate samples. The in vivo results are also represented as mean ± SEM from 5 mice per group. The results were considered statistically significant at a value of p < 0.05. 3. Results 3.1. Regulatory effect of Atr on degranulation of mast cells First, we investigated whether Atr would have a regulatory effect on degranulation of mast cells. We measured calcium and granule-associated mediators levels, such as tryptase and histamine in RPMCs and HMC-1 cells. As shown in Fig. 2A, Atr dosedependently inhibited intracellular calcium level in compound 48/80-stimulated RPMCs (p < 0.05). Atr significantly inhibited tryptase and histamine releases from the activated RPMCs (p < 0.05; Fig. 2B and C). And we observed whether Atr would affect morphology of the activated RPMCs. Atr significantly decreased the number of degranulated RPMCs (p < 0.05; Fig 2D). Further, we examined whether Atr would regulate the activity of p56lck tyrosine kinase as compared to that of Dam which is a selective inhibitor of p56lck tyrosine kinase [26]. Atr suppressed the activity of p56lck tyrosine kinase increased by compound 48/80 similarly to an

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Fig. 2. Regulatory effect of Atr on degranulation of RPMCs. RPMCs were treated with Atr, KMP6, Ketotifen, or Bapta-AM and stimulated with compound 48/80 (6 mg/mL). (A) The intracellular calcium levels were determined using the fluorescence. (B) Tryptase level was determined using a mast cell degranulation assay kit. (C) Histamine level was measured using o-phthalaldehyde spectrofluorometric method. (D) The number of degranulated RPMCs was expressed as the percentage of degranulated RPMCs. Images show representative of degranulated RPMCs. Data were expressed as mean ± SEM of three independent experiments with duplicate samples. Atr, atractylone; Blank, inactivated RPMCs. #p < 0.05 vs inactivated RPMCs. *p < 0.05 vs compound 48/80-stimulated RPMCs.

inhibitory effect of Dam (p < 0.05; Fig 3A). And we confirmed the inhibitory effect of Dam with Atr and KMP6 on histamine release from the activated RPMCs (p < 0.05; Fig 3B). Next, we investigated the regulatory effect of Atr on degranulation of PMACI-stimulated HMC-1 cells. Atr significantly decreased calcium level in the activated HMC-1 cells similarly to an inhibitory effect of Bapta-AM on the intracellular calcium level (p < 0.05; Fig. 4A). Also, Atr significantly decreased tryptase and histamine releases from the

activated HMC-1 cells (p < 0.05; Fig. 4B and C). Furthermore, Atr decreased HDC activity and expression in the activated HMC-1 cells (Fig. 4D and E). DEX as a reference drug [27] inhibited HDC activity and expression in the activated HMC-1 cells in this study (p < 0.05; Fig. 4D and E). KMP6 (0.1 mg/mL) or ketotifen also significantly inhibited intracellular calcium, tryptase, histamine levels in the activated RPMCs and HMC-1 cells (p < 0.05; Figs. 2AeC and 4A-C). Atr did not affect the tryptase release, histamine release, HDC

Fig. 3. Regulatory effect of Atr on the activation of p56lck tyrosine kinase. RPMCs were treated with Atr, KMP6, or Dam and stimulated with compound 48/80 (6 mg/mL). (A) The enzymatic activity of p56lck tyrosine kinase was examined using a p56lck tyrosine kinase assay kit. (B) Histamine level was measured using o-phthalaldehyde spectrofluorometric method. Atr, Atractylone; Dam, damnacanthal. #p < 0.05 vs inactivated RPMCs. *p < 0.05 vs compound 48/80-stimulated RPMCs.

N.-R. Han et al. / Chemico-Biological Interactions 258 (2016) 59e68 Fig. 4. Regulatory effect of Atr on histamine release from activated HMC-1 cells. HMC-1 were treated with Atr, KMP6, Ketotifen, or Bapta-AM and then stimulated with PMACI. (A) The intracellular calcium levels were determined using the fluorescence. (B) Tryptase level was determined using a mast cell degranulation assay kit. (C) Histamine level was measured using o-phthalaldehyde spectrofluorometric method. (D) The activity of HDC was determined using HDC assay as described in the Materials and method section. (E) The expression of HDC was analyzed with Western blot analysis. Data were expressed as mean ± SEM of three independent experiments with duplicate samples. PMACI, PMA plus A23187; Atr, Atractylone; DEX, dexamethasone. #P < 0.05 vs inactivated HMC-1 cells. *P < 0.05 vs PMACI-stimulated HMC-1 cells.

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activity, and HDC expression in the inactivated RPMCs or HMC1 cells (Fig. 2B and C, 4B-E). And Atr did not affect the intracellular calcium level in the inactivated RPMCs and HMC-1 cells (data not shown). 3.2. Regulatory effect of Atr on SCF-induced F-actin formation To further determine whether Atr would regulate morphological change of RPMCs, RPMCs were treated with Atr and then stimulated with SCF. We observed the morphological alterations of

the RPMCs for 4 days. The stimulation with SCF significantly induced the morphological alterations, whereas Atr, KMP6, or DEX decreased this morphological alteration (p < 0.05; Fig. 5A). Next, we investigated whether Atr would suppress SCF-induced F-actin formation. As shown in Fig. 5B, Atr, KMP6, or DEX inhibited SCFinduced F-actin formation. 3.3. Regulatory effect of Atr on PCA Given an inhibitory effect of Atr on the degranulation of RPMCs

Fig. 5. Regulatory effect of Atr on SCF-induced F-actin formation. (A) RPMCs (3  104) were treated with Atr, KMP6, or DEX for 1 h and then stimulated with SCF (50 ng/mL) for 4 days. Morphological alteration was evaluated by counting the number of RMPCs. Images show representative of morphological alteration on 4th day. Data were expressed as mean ± SEM of three independent experiments with duplicate samples. (B) RPMCs (3  104) were treated with Atr, KMP6, or DEX for 1 h and then stimulated with SCF (50 ng/mL) for 1 h. RPMCs were stained with NBD-phallacidin. F-actin was visualized using a confocal laser scanning microscope. Each data was expressed as mean ± SEM from three separate experiments with duplicate samples. Atr, atractylone; DEX, dexamethasone; Blank, inactivated RPMCs. #p < 0.05 vs inactivated RPMCs; *p < 0.05 vs SCF-stimulated RPMCs.

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Fig. 6. Regulatory effect of Atr on PCA. Atr, KMP6, or Ketotifen was orally administered 1 h before the injection with DNP-HSA. Each amount of dye was measured at 620 nm in an ELISA reader and expressed as the mean ± SEM. n ¼ 5/group. DNP-HSA, dinitrophenyl-human serum albumin; Atr, Atractylone. *P < 0.05 vs PCA-induced mice.

and HMC-1 cells, we investigated whether Atr could inhibit PCA. As a result, Atr significantly inhibited the PCA reaction (p < 0.05; Fig. 6). Also, Atr significantly reduced histamine, IgE, IL-1b, IL-4, IL5, IL-6, VEGF, and IL-13 levels in the serum under PCA (p < 0.05; Fig. 7). However, Atr did not decrease TNF-a level in the serum under PCA. KMP6 (0.1 g/kg) also significantly inhibited the histamine, IgE, IL-1b, IL-4, IL-5, IL-6, VEGF, and IL-13 levels in the serum under PCA (p < 0.05; Fig. 7). Ketotifen significantly inhibited the histamine, IgE, IL-1b, VEGF, and IL-13 levels (p < 0.05; Fig. 7). 3.4. Regulatory effect of Atr on systemic anaphylaxis To evaluate whether Atr would regulate systemic anaphylaxis, Atr was orally administered to mice and then compound 48/80 was injected intraperitoneally. As shown in Table 1, Atr inhibited compound 48/80-induced mortality. KMP6 or ketotifen also suppressed compound 48/80-induced mortality (Table 1). 3.5. Regulatory effect of Atr on compound 48/80-induced ear swelling response To further elucidate the inhibitory effect of Atr on allergic reactions, we examined ear swelling response in the mice treated with Atr. Atr significantly suppressed the ear swelling response to compound 48/80 (p < 0.05; Fig. 8). KMP6 or ketotifen also significantly inhibited the ear swelling response (p < 0.05; Fig. 8). 4. Discussion The compounds of KMP6, Magnolia officinale, Citrus sunki, Zingiber officinale, Glycyrrhiza uralensis, and Zizyphus jujube had antiinflammatory or anti-allergic effects [28e32]. Atractylodes japonica which is a main component of KMP6 was also reported to have anti-allergic actions on atopic dermatitis [33]. KMP6 has various phytochemical constituents, such as terpenoids which have been used for therapeutic purposes as anti-inflammatory agents [34]. Citrus contains various terpenoid constituents (limonoids), such as nomilin and limonene which have an anti-inflammatory effect [35e37]. Magnolia bark also contains terpenoids as principal substantial constituents [28]. Zingiber officinale contains terpenoid constituents, such as gingerol which has an antiinflammatory effect [38]. Atractylenolide III, an active terpenoid component of Atractylodes japonica showed an anti-inflammatory effect by suppressing the releases of TNF-a and IL-6 through the

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blockade of nuclear factor-kB/mitogen-activated protein kinasesignaling pathways [39]. Atr, a terpenoid component, possessed an inhibitory effect on tumor cell growth [40] and a protective effect against oxidative stress [16]. And Atr showed an inhibitory activity against ear inflammation in mice [41]. Thus, we hypothesized that Atr would have a regulatory effect on allergic reactions. In our research, we observed the regulatory effect of Atr on allergic mediators in vitro models, compound 48/80-stimulated RPMCs, PMACI-stimulated HMC-1 cells, and SCF-stimulated RPMCs and in vivo models, IgE-mediated PCA, compound 48/80-induced systemic anaphylaxis, and ear swelling. Atr was determined to have an anti-allergic effect by suppressing mast cell degranulation or stabilizing mast cell. Interestingly, KMP6 (0.1 mg/ml) suppressed the allergic reactions more than Atr, depending on the measured factors, such as the number of degranulated RPMCs and SCF-induced morphological alteration. They may result from interactions between components of KMP6 including terpenoids. However, there were also the other factors which do not show notable difference between groups of Atr and KMP6 (0.1 mg/ml). In addition, our previous study reported that KMP6 (1 mg/ml) inhibited the allergic reactions more than hesperidin, a component of KMP6, depending on the measured factors [19]. Thus, further study is required to elucidate synergistic effects between the components of KMP6 on allergic reactions. Mast cell degranulation can initiate releases of vasoactive and proinflammatory mediators, such as histamine, tryptase, IL-6, and VEGF via cross-linking of IgE-FcεRI [42]. Compound 48/80 (a mixture of polymers derived from N-methyl-p-methoxy-phenylethylamine) is known to be a potent mast cell secretagogue [43] and induce perturbation of membrane with increased permeability of membrane [44]. And compound 48/80 activates phospholipase D (PLD) and induces tyrosine phosphorylation in mast cells [45,46]. The activation of PLD, protein kinase C (PKC), and tyrosine kinase, and calcium mobilization are critical signals for degranulation of mast cells [46,47]. PMA and A23187 used in the activation of HMC-1 cells is a highly potent PKC activator and calcium ionophore respectively. The intracellular calcium pathways are an important for the degranulation of mast cells [48]. A mast cell stabilizer, ketotifen inhibited intracellular calcium level and the degranulation of mast cells [49,50]. In addition, ketotifen is widely used in the treatment of allergic disorders [51]. In this study, intracellular calcium level was increased in the activated mast cells. Similarly to the inhibitory effect of ketotifen, Atr inhibited intracellular calcium, tryptase, and histamine levels in the activated mast cells. In addition, Dam, an inhibitor of tyrosine kinase, was reported to inhibit mast cell-mediated anaphylactic reactions [24]. Atr suppressed the activation of tyrosine kinase similarly to the inhibitory effect of Dam. Also, Atr inhibited the compound 48/80induced systemic anaphylaxis and ear swelling. In addition, Atr suppressed an increase in F-actin content in the SCF-stimulated RPMCs. Hence, we can assume that Atr might regulate the mast cell degranulation and inhibit allergic reactions via downregulation of tyrosine kinase. Histamine is a marker of mast cell degranulation [52]. HDC is a unique enzyme which catalyzes histamine formation from L-histidine. HDC can be a potential target for therapeutical intervention of many allergic inflammatory diseases [53]. DEX inhibited HDC activity and expression and blocked development of nasal hypersensitivity [27]. In this study, PMACI increased the HDC activity and expression in the activated HMC-1 cells. Similarly to the inhibitory effect of DEX, Atr inhibited HDC activity and expression in the activated HMC-1 cells. Thus, we speculate that Atr inhibits the mast cells degranulation, down-regulating histamine synthesis. The IgE and allergen-dependent mast cells activation via Fc3RI promotes immediate release of histamine or synthesized cytokines,

66 N.-R. Han et al. / Chemico-Biological Interactions 258 (2016) 59e68 Fig. 7. Regulatory effect of Atr on levels of histamine and cytokines under PCA reaction. Serum was obtained from heart. (A) Serum histamine level was measured using o-phthalaldehyde spectrofluorometric method. The levels of serum (B) IgE, (C) IL-1b, (D) IL-4, (E) IL-5, (F) IL-6, (G) VEGF, (H) IL-13, and (I) TNF-a were measured with ELISA method. Data were expressed as mean ± SEM. n ¼ 5/group. DNP-HSA, dinitrophenyl-human serum albumin; Atr, Atractylone. #P < 0.05 vs normal mice. *P < 0.05 vs PCA-induced mice.

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Acknowledgments

Table 1 Regulatory effect of Atr on systemic anaphylaxis. Treatment

Dose

Compound 48/80

Mortality (%)

None (saline) Atr Atr Atr KMP6 KMP6 Ketotifen

 0.25 (mg/kg) 2.5 (mg/kg) 25 (mg/kg) 50 (mg/kg) 100 (mg/kg) 2 (mg/kg)

þ þ þ þ þ þ þ

100 50 60 20 70 40 0

The groups of mice were orally administered with saline, Atr, KMP6, or ketotifen 1 h before compound 48/80 (8 mg/kg) injection. Mortality (%) is expressed as ratio of number of dead mice to total number of mice. Data were expressed as mean. n ¼ 10/ group. Atr; Atractylone.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2015R1D1A1A01056607). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.08.015 References

Fig. 8. Regulatory effect of Atr on compound 48/80-induced ear swelling response. Atr, KMP6, or ketotifen was orally administered 1 h before compound 48/80 injection. Data were expressed as mean ± SEM. n ¼ 5/group. Atr, Atractylone. *P < 0.05 vs ear swelling-induced mice.

which plays a critical role in the pathogenesis of allergic diseases [54]. The released histamine severely increases vascular permeability. PCA which is characterized by increased permeability of cutaneous vessels is an immediate dermal reaction via an allergenIgE interaction [55]. Thus, PCA has widely been used as a tool to investigate sensitivity to allergens and agent efficacy in inhibiting allergic reaction. IL-1b, IL-4, IL-5, IL-6, VEGF or IL-13 was increased in serum of patients with acute allergic reactions [56e58]. In this study, the interaction with DNP-HSA and anti-DNP-IgE induced PCA reaction and the releases of histamine, IL-1b, IL-4, IL-5, IL-6, IL-13 or VEGF in the serum. However, Atr-treated mice were protected from this IgE-induced PCA reaction, inhibiting histamine and inflammatory cytokines levels. Thus, the treatment with Atr enables to inhibit mast cell-mediated allergic reactions. In summary, the above findings suggest that Atr may contribute to ameliorate mast cell-mediated allergic reactions. The treatment with Atr might serve a protective role in anaphylaxis or ear swelling, possibly by inhibiting the degranulation of mast cells. Nonetheless, further research is required to fully elucidate the role of Atr in diverse allergy models. As the first to elaborate the regulatory effect of Atr on mast cell-mediated allergic reactions, this study could provide knowledge regarding the anti-allergic effect of Atr.

Conflict of interest statement None declared.

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