Asiaticoside Mitigates the Allergic Inflammation by Abrogating the

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Asiaticoside Mitigates the Allergic Inflammation by Abrogating the Degranulation of Mast Cells Jing Zhi Jiang,†,⊥ Jing Ye,†,⊥ Guang Yu Jin,‡,⊥ Hong Mei Piao,‡ Hong Cui,† Ming Yu Zheng,§ Jin Shi Yang,§ Nan Che,† Yun Ho Choi,∥ Liang Chang Li,*,† and Guang Hai Yan*,† †

Department of Anatomy, Histology and Embryology, Medical College of Yanbian University, Yanji 133002, Jilin, China Department of Respiratory Medicine, Yanbian University Hospital, Yanji 133000, Jilin China § College of Pharmacy, Yanbian University, Yanji 133002, Jilin, China ∥ Department of Anatomy, Medical School of Institute of Medical Sciences, Chonbuk National University, Jeonju, Jeonbuk 561-756, Republic of Korea ‡

ABSTRACT: The effects of asiaticoside (AS) on allergic responses mediated by mast cells were investigated. AS showed no obvious cytotoxicity on RPMCs (rat peritoneal mast cells). AS reduced the intracellular calcium in RPMCs and deprived the histamine release and degranulation. AS also decreased the generation of antigen-induced tumor necrosis factor α, interleukin (IL)-4, IL-8, and IL-1β in RBL-2H3 cells sensitized by IgE. The suppression of AS on pro-inflammatory cytokines was related with the activation of the intracellular FcεRI and the inhibition of the nuclear factor-κB signaling pathway. In addition, AS disabled the phosphorylation of antigen-induced Syk, Lyn, Gab2, and PLCγ1, thus suppressing the downstream Akt phosphorylation and MAPKs pathways. It also increased HO-1 and Nrf2 expression time dependently. In summary, we demonstrate that AS suppresses the allergic inflammation mediated by mast cells and this effect might be mediated by FcεRI-dependent signaling pathways. KEYWORDS: asiaticoside, allergic inflammation, degranulation, mast cell

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INTRODUCTION Type I allergy is an immune disorder which can result in immunoglobulin E (IgE) production. After inducement by allergens, mast cells can release inflammatory mediators, which play important roles in IgE-mediated allergic diseases.1,2 Thus, allergic inflammation is mainly mediated by mast cells. The cross-link between antigen-binding IgE and FcεRI could result in the FcεRI aggregation, triggering the intracellular signaling process to phosphorylate the kinases Lyn and Fyn of the Src family. And then, the downstream Syk and other tyrosine kinases are activated to mobilize the internal calcium ion.3−5 Inflammatory mediators are then synthesized and the pathways of phosphatidylinositol 3-kinase (PI3K) and Akt, mitogenactivated protein kinase (MAPKs), and nuclear factor-κB (NF-κB) are activated.6,7 The inflammatory mediators released by the mast cells mainly include histamine, eicosanoids, proteases, and other chemotactic and pro-inflammatory cytokines.8,9 The histamine produced by the activated mast cells is the major factor in acute allergic responses. Histamine could induce increased permeability and vasodilation of vessels, which will result in edema, hypothermia, and leukocyte recruitment.10 The chemotactic and pro-inflammatory cytokines are released to attract the basophils and neutrophils, which will mediate the allergic inflammation in the late phase.11 So, the mast cells may be used as potential target for the development of antiallergic drugs. Centella asiatica, which belongs to the Apiaceae family, is a tropical plant originated from Southeast Asian countries.12 In Indonesia and Malaysia, Centella asiatica is eaten fresh as a vegetable.13 The raw Centella asiatica is eaten along with the © XXXX American Chemical Society

main meal or serves as an appetizer. It is also eaten after being cooked. The coconut milk, shredded coconut, and sometimes sweet potatoes are always added while cooking Centella asiatica. It is employed as a tonic and is available at some markets as a ready-made juice.14 Asiaticoside (AS), extracted from Centella asiatica, possesses antioxidant and anti-inflammatory functions in several experimental animal models.15 However, the roles of AS in allergic response are unclear. Here, we investigated the effects and the mechanisms of AS on mast cell mediated allergic response.

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MATERIALS AND METHODS

Animals. The 7-week-old male BALB/c mice (n = 100) and male Sprague−Dawley rats (n = 20) were obtained from the House section of Yanbian University Health Science Center (YanJi, China). All the mice were kept under the standard conditions. The guidelines of the Institutional Animal Care and Use Committee of Yanbian University School of Medical Sciences were followed during the exprements. Cell Culture and Treatment. RBL-2H3 cells were cultured in DMEM (Gibco, Grand Island, NY) and 10% fetal bovine serum (Gibco, Grand Island, NY) and 100 U/ml penicillin G, 100 μg/mL streptomycin at 37 °C in 5% CO2. The cells were divided into control group, IgE+Ag group and IgE+Ag+AS group, respectively. Cells in control group were normally cultured without any drug treatment. Cells in IgE+Ag group were treated with 10 μg/mL anti-DNP IgE (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for 6 h and Received: April 24, 2017 Revised: August 28, 2017 Accepted: August 30, 2017

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DOI: 10.1021/acs.jafc.7b01590 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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challenged with DNP-HSA (Sigma) (100 ng/mL) for 10 min. In IgE+Ag+AS group, besides IgE treatment, AS (5, 10, and 20 μM) (with purity of 98%; TW Reagent Co., Ltd., Shanghai, China) was added for incubation for 30 min at 37 °C before challenging with DNP-HSA (100 ng/mL). Passive Cutaneous Anaphylaxis (PCA) Test. PCA mediated by anti- DNP IgE was performed as described.16 Briefly, mice were divided into control group, IgE+Ag group and IgE+Ag+AS group, with 10 mice in each group. Mice in the control group received phosphatebuffered saline (PBS) of equal volume. Mice in IgE+Ag group were sensitized by 200 ng anti-DNP IgE in 20 μL PBS intradermally in the dorsal skin on the right, and a sham PBS injection were given in the left dorsal skin. At 24 h later, every individual mouse was injected via tail vein with 0.1 mg antigen (DNP-HSA) and 1% Evans blue (Sigma). In IgE+Ag+AS group, at 1 h before the antigen challenge, AS (10, 20, 40 mg/kg of body weight, respectively) was administered orally. Then, after 30 min, the mice were sacrificed by terminal anesthesia. The weight of the pigment area in the remove skin at the injection site was measured. After incubating the removed skin in formamide for 24 h at 55 °C, the extravasated Evans blue dye was extracted. The amount of dye absorbance was measurement at 620 nm wavelength with a Spectra MAX PLUS spectrophotometer (Molecular Devices, CA, U.S.A.). Ear Swelling Response in Mice. Mice were assigned into IgE+Ag group, IgE+Ag+AS group, and control group, with 10 mice in each group. Mice in control group received 20 μL PBS. Mice in IgE+Ag group were injected intradermally with 200 ng of anti-DNP IgE in the ear. After 24 h, the mice received 0.1 mg DNP-HAS through tail vein injection. For mice in IgE+Ag+AS group, 1 h before the antigen challenge, AS (10, 20, 40 mg/kg of body weight) was orally administered. One hour after the challenge, mild anesthesia was induced in mice and a digital micrometer (Mitutoyo, No. 7326, Japan) was used to measure the thickness of the ear. Histology Analysis and Mast Cell Count. After measurement of the thickness, the ears were removed. After fixation and embedment, the ears were cut into sections (5 μm). The hematoxylin and eosin and toluidine blue (Sigma) staining was conducted according to routine procedure. The number of mast cells was counted in five fields randomly selected under 100× magnification. RPMCs Preparation and Treatment. Rat peritoneal mast cells (RPMCs) were collected according to previous report.17 Rats were anesthetized with 10 mL calcium-free HEPES-Tyrode buffer (Sigma) in the peritoneal cavity After opening the peritoneal cavity, the fluid containing peritoneal cells was collected. RPMCs were isolated with Percoll (Pharmacia, Uppsala, Sweden) density centrifugation. The isolated RPMCs (1 × 106 cells/ml) were then resuspended in HEPES-Tyrode buffer. RPMCs were divided into control group, IgE+Ag group and IgE+Ag+AS group. Cells in control group were normally cultured without any drug treatment. Cells in IgE+Ag group were treated with 10 μg/mL anti-DNP IgE for 6 h and challenged with DNP-HSA (100 ng/mL) for 10 min. In IgE+Ag+AS group, besides IgE treatment for 6 h, cells were then preincubated with AS (5, 10, and 20 μM) for 30 min at 37 °C before the incubation with DNP-HSA (100 ng/mL). MTT Assay. RBL-2H3 cells or RPMCs (2 × 104/well in 96-well plates) were pretreated with AS for 24 h. Then, 1 mg/mL MTT was added into cells for incubation at 37 °C. And 2 h later, DMSO was added. The absorbance was detected with a Spectra MAX PLUS spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at 570 nm wavelength. Histamine Release and Microscopic Observation. The radio enzymatic method was used to detect histamine release by RPMCs.14 Briefly, after cell grouping and challenging with DNP-HSA (100 ng/mL) for 10 min, the supernatant was collected by 150 g centrifugation at 4 °C for 10 min. Then the concentration of histamine was measured. RPMCs (1 × 106 cells/ml) were observed and photographed after resuspension in HEPES-Tyrode buffer,. 45 Ca Uptake Measurement. The calcium uptake by RPMCs was detected according to the previously described procedure.18 In brief, RPMCs were resuspended in HEPES-Tyrode buffer containing

Ca (1.5 mCi/mL, PerkinElmer Life Sciences, MA, USA). After incubation at 4 °C for 10 min, RPMCs were then grouped and treated as above-described. After centrifugation, 10% Triton X-100 was added to lyse cells. The radioactivity was measured with a Liquid Scintillation Analyzer (A Canberra Company, Australia). RT-qPCR. Total RNA was collected from treated RBL-2H3 cells using a Trizol extraction kit (Invitrogen Inc., Carlsbad, CA). The Prime Script RT-PCR kit (Takara, Dalian, China) was used for RNA reverse transcription into cDNA. The primers used for RT-qPCR were: TNF-α forward: 5′-GATCGGTCCCAACAAGGAGG-3′, reverse: 5′-GTGAGGAGCACATAGTCGGG-3′; IL-4 forward: 5′- TCCACGGATGTAACGACAGC-3′, reverse: 5′- TCATTCACGGTGCAGCTTCT-3′; IL-1β forward: 5′- TTGAGTCTGCACAGTTCCCC-3′, reverse: 5′-GTCCTGGGGAAGGCATTAGG-3′; IL-8 forward: 5′-TGGCCAGAGAAAGAAGTGCC-3′, reverse: 5′-TGTCTTCAATCCATCCCAGAGC-3′ and GAPDH forward: 5′- AGACAGCCGCATCTTCTTGT-3′, reverse: 5′-CTCGTGGTTCACACCCATCA-3′. RT-qPCR was performed on the Chromo 4 instrument (Bio-Rad, Hercules, CA). The PCR procedures were: 95 °C denaturation for 10 min, and 40 cycles of 95 °C denaturation for 15 s, 60 °C annealing for 1 min and 72 °C extension for 40 s. The amplified products underwent electrophoresis and were filmed by the Gel Doc XR system (Bio-Rad, CA, USA). ELISA. The supernatant of treated RBL-2H3 cells was collected. ELISA was performed using TNF-α, IL-4, IL-1β ELISA kits (Abcam, Cambridge, UK) and an IL-8 ELISA kit (Cusabio, Wuhan, China), respectively. The absorbance at 450 nm wavelength was measured. Western Blot. The nuclear and cytosolic proteins were isolated from treated RBL-2H3 cells.19 Then, proteins were separated on SDS-PAGE and transferred to PVDF membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). After blockage in 5% nonfat milk for 1 h, the primary antibodies were used and incubation was performed at 4 °C overnight. The primary antibodies of anti-NF-κB p65, anti-IκBα, antiphospho-IκBα, anti-JNK, antiphospho-JNK, antiPLCγ1, anti-PARP and antiactin were all purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). The primary antibodies of anti-Syk, antiphospho-Syk, anti-Lyn, antiphospho-Lyn, anti-Gab2, antiphosphoGab2, anti-Akt, antiphospho-Akt, anti-p38 MAPK, antiphospho-p38 MAPK, anti-ERK, antiphospho-ERK, anti-Nrf-2, anti-HO-1 were purchased from Cell signaling Technology (Beverly, MA, USA). The secondary antibody for antirabbit horseradish peroxidase conjugatedIgG was purchased from Santa Cruz, CA, USA. Densitometric scanning using the Gel Doc XR system (Bio-Rad, CA, USA) was used to analyze the immuno-reactive and phosphorylation signals. Statistical Analyses. The data were showed as mean ± SEM. Prism 5.0 (GraphPad Software, San Diego, CA) was used to analyze the statistical differences. One-way analysis of variance followed by Tukey’s test for post hoc analysis was used. A P value lower than 0. 05 indicated a significant difference.

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RESULTS AND DISCUSSION AS Shows Antiallergy Effect on Antigen-Mediated PCA. The antiallergy effect of AS was evaluated by anlyzing the local extravasation of Evans blue dye in PCA mice model. AS was orally administered 1 h before antigen challenge. The level of dye extravasation was significantly higher in IgE+Ag group than control group. AS (10, 20, 40 mg/kg of body weight) inhibited the dye extravasation dose dependently (Table 1). AS at doses of 20 and 40 mg/kg significantly reduced anti-DNP IgE-induced dye extravasation compared with IgE+Ag group. AS Relieves the Antigen-Induced Ear Histology and Ear Swelling. To investigate the effect of AS on anti-DNP IgE-induced ear histology, the toluidine blue and hematoxylin and eosin staining was performed. Compared with the control group, anti-DNP IgE-induced mice in the IgE+Ag group showed a remarkable thickened ear, hypodermis edema, and inflammatory cells infiltration into the ear skin dermis (Figure 1A). However, the administration of AS inhibited the B

DOI: 10.1021/acs.jafc.7b01590 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Normal RPMC cells were round with a regular surface and fine granules (Figure 2B). However, after treatment with IgE+Ag, RPMC cells were degranulated, showing cytoplasmic vacuoles, cell swelling, extruded granules, and an irregular surface. Preincubation with AS inhibited the degranulation of RPMCs. However, the cell size was a little larger than that of the normal RPMC cells (Figure 2B). The histamine release was also evaluated. AS was preincubated at a concentration ranging from 5 to 20 μM. As shown in Figure 2C, AS concentration-dependently inhibited anti-DNP IgE-mediated histamine release from RPMC. To reveal the mechanisms underlying the suppressive effects of AS on histamine release, we tested the calcium uptake. As shown in Figure 2D, the intracellular calcium uptake in the IgE+Ag group was significantly increased. However, this increase was significantly inhibited by AS (10 and 20 μM). AS Inhibits the Expression of Inflammatory Cytokine. Mast cells activation could stimulate cytokines release. Thus, we tested whether the release of TNF-α, IL-8, IL-1β, and IL-4 is affected by AS in RBL-2H3 cells. As in Figure 3A, the TNF-α, IL-1β, IL-4, and IL-8 mRNA levels were high in the IgE+Ag group. However, AS administration greatly inhibited these mRNA levels’ dose dependently. Then, we investigated whether AS suppressed the secretion of all cytokines in RBL-2H3 cell supernatant. Inconsistent with the RT-PCR results, ELISA revealed that the cytokines’ levels were significantly elevated in the IgE+Ag group compared with the control group; however, the cytokines secretions were all inhibited by AS administration at the concentrations 10 and 20 μM in RBL-2H3 cells (Figure 3B). Therefore, AS may play an inhibitive role in the inflammatory cytokine secretion by mast cells. AS Down-Regulates the FcεRI-Mediated Signaling in Antigen-Induced Mast Cells. To anticipate the potential

Table 1. Effects of Asiaticoside on Anti-DNP IgE-Mediated Passive Cutaneous Anaphylaxis in Micea Group Control IgE+Ag IgE+Ag+AS

10 (mg/kg BW) 20 (mg/kg BW) 40 (mg/kg BW)

Amount of Evans blue (μg/g) 48.59 254.69 226.51 172.42 151.86

± ± ± ± ±

5.26 10.59* 9.56* 10.25*# 7.68*##

a Note: Each data value represents the mean ± SEM of five independent experiments. *p < 0.05 vs control group. #p < 0.05. ##p < 0.01 vs IgE+Ag group. BW, body weight.

above pathological changes (Figure 1A). Consistent with these results, AS administration also relieved anti-DNP IgE-induced ear swelling responses and significantly decreased the thickness of the ear (Figure 1C). Compared with the control mice, anti-DNP IgE-induced mice in the IgE+Ag group markedly increased the mast cell number in the ear. However, AS administration showed no obvious effect on mast cell number (Figure 1B and 1D). These results suggested that AS inhibited inflammatory responses including activation of mast cell and inflammatory swelling in antigen-induced allergic responses. AS Decreases the Release of Histamine and the Uptake of the Intracellular Calcium. To check the effects of AS on the cell viability, MTT assay was conducted. RPMCs and RBL-2H3 cells were incubated with indicated concentrations (1−100 μM) of AS for 24 h. As shown in Figure 2A, different concentrations of AS did not obviously affect the cell viability of RBL-2H3 cells or RPMCs. To investigate the effect of AS on the degranulation of the mast cell, the morphology of RPMCs was photographed.

Figure 1. Effect of asiaticoside on Anti-DNP IgE-mediated ear swelling response in mice. Ear swelling response was induced in mice. Mice were divided into the control group, IgE+Ag group, and IgE+Ag+AS group. (A and B) Hematoxylin and eosin and toluidine blue staining for ear sections (magnification ×200). (C) The ear thickness was measured with a dial thickness gauge. (D) The number of mast cells at the dermis was counted after toluidine blue staining. Each data value represents the mean ± SEM of five independent experiments. # p < 0.05 vs control group; *p < 0.05. **p < 0.01 vs IgE+Ag group. C

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Figure 2. Effect of asiaticoside on cells viability and the degranulation of rat peritoneal mast cells (RPMCs). (A) The purified RPMCs and RBL-2H3 cells were treated with various concentrations of asiaticoside. The cell viability was detected by MTT assay. (B) Effect of asiaticoside on anti-DNP IgE-mediated degranulation of RPMCs (magnification, ×1000). RPMC cells were divided into the control group, IgE+Ag group, and IgE+Ag+AS group. (C and D) The histamine release and calcium uptake were measured by the radioenzymatic method. Each data value represents the mean ± SEM of five independent experiments. #p < 0.05 vs control group; *p < 0.05; **p < 0.01 vs IgE+Ag group.

Figure 3. Effect of asiaticoside on pro-inflammatory cytokines in RBL-2H3 cells. RBL-2H3 cells were divided into the control group, IgE+Ag group, and IgE+Ag+AS group. Cells were sensitized with 10 μg/mL anti-DNP IgE for 6 h and challenged with 100 ng/mL DNP-HAS in the absence or presence of asiaticoside (5, 10, and 20 μM). (A) The mRNA expression levels of cytokines were measured by RT-PCR. (B) The levels of cytokines in the culture supernatant were measured by ELISA. Each data value represents the mean ± SEM of five independent experiments. #p < 0.05 vs control group; *p < 0.05 vs IgE+Ag group. D

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Figure 4. Effect of asiaticoside on the activating phosphorylation of Syk- and Syk-mediated downstream molecules. RBL-2H3 cells were divided into the control group, IgE+Ag group, and IgE+Ag+AS group. RBL-2H3 cells were sensitized with 10 g/mL anti-DNP IgE for 6 h and then challenged with 100 ng/mL DNP-HAS in the absence or presence of asiaticoside (5, 10, and 20 μM). Western blot was performed. Each data value represents the mean ± SEM of five independent experiments. #p < 0.05 vs the control group; *p < 0.05 vs IgE+Ag group.

Figure 5. Effect of asiaticoside on the activation of AKT and MAPKs signaling. RBL-2H3 cells were divided into control group, IgE+Ag group, and IgE+Ag+AS group. RBL-2H3 cells were sensitized with 10 μg/mL anti-DNP IgE for 6 h and challenged with 100 ng/mL DNP-HAS in the absence or presence of asiaticoside (5, 10, and 20 μM). Western blot was performed. Each data value represents the mean ± SEM of five independent experiments. #p < 0.05 vs control group; *p < 0.05 vs IgE+Ag group.

molecular target on which AS functioned, the influences of AS on signaling pathways mediated by FcεRI in RBL-2H3 cells were investigated. The phosphorylation of Syk, Lyn, and Gab2 (Figure 4), key proteins of the signaling pathway, were suppressed by AS. Furthermore, it also inhibited the phosphorylation of the downstream molecular PLC-γ1. The changes of AKT, p38, JNK and ERK after AS treatment were detected by Western blotting. The phosphorylation of p38, ERK, and JNK in MAPKs pathways was increased significantly in the IgE+Ag group compared to in the control group (Figure 5). However, these pathways were suppressed by AS dose dependently. AS Down-Regulates the Activation of NF-κB Translocation by Nrf2/HO-1 Signaling. To reveal the mechanisms of the inhibitory role of AS in inflammatory cytokine release, Western blotting was then conducted to evaluate NF-κB translocation. As in Figure 6, AS elevated the level of NF-κB p65 in the cytosol and decreased its level in the nuclear. Besides, AS markedly suppressed the NF-κB p65 transfer into the nuclear. Then, the level of IκBα after AS treatment was analyzed. We found that AS significantly blocked the antigen-induced

phosphorylation of IκBα (Figure 6). Several studies had shown that Nrf2/HO-1 could prevent the release of inflammatory cytokine.20 Then, the role of AS in Nrf2 activation and HO-1 expression was studied. Our results suggested that AS at a large degree activated Nrf2 and promoted HO-1 expression in antigen-induced mast cells time dependently (Figure 7). Mast cells have long-living capacity and are tissue-resident cells. And they are originated from hematopoietic stem cells at the interface. Thus, mast cells can be triggered by foreign antigens in the environment and induce the inflammatory responses and production of inflammatory mediators,21 thus playing critical roles in allergy mediated inflammation.22 The localized allergic reactions mediated by mast cells can be evaluated with the PCA animal model in vivo.23 Our results showed that AS significantly inhibited the leakage of Evans blue in PCA mice and markedly decreased the thickness of ears in the antigen-induced allergy responses. However, AS attenuated the localized inflammatory response without disturbing the numbers of mast cells. Together, these data reveal that the suppressive effects of AS on the activation of mast cells may not be caused by the decreased quantity of mast cells. E

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Figure 6. Effect of asiaticoside on NF-κB activation in RBL-2H3 cells. RBL-2H3 cells were divided into the control group, IgE+Ag group, and IgE+Ag+AS group. RBL-2H3 cells were sensitized with 10 μg/mL anti-DNP IgE for 6 h and challenged with 100 ng/mL DNP-HAS in the absence or presence of asiaticoside (5, 10, and 20 μM). Western blot was performed. Each data value represents the mean ± SEM of five independent experiments. #p < 0.05 vs the control group; *p < 0.05 vs IgE+Ag group.

Figure 7. Effect of asiaticoside on the proteins expression of HO-1 and Nrf2 in RBL-2H3 cells. RBL-2H3 cells were sensitized with 10 μg/mL anti-DNP IgE for 6 h and then challenged with 100 ng/mL DNP-HAS in the presence of 20 μM asiaticoside at the indicated times (0, 0.5, 1, 1.5, 2 h) for Nrf2 and (0, 3, 6, 12, 18 h) for HO-1. (A and B) Western blot was performed. Each data value represents the mean ± SEM of five independent experiments. *p < 0.05 vs 0 h which was sensitized with 10 μg/mL anti-DNP IgE for 6 h and challenged with 100 ng/mL DNP-HAS in the absence of 20 μM asiaticoside.

It is known that mast cells are activated by IgE and then secret various mediators, which play important roles in allergy. 24 Therefore, it is important to inhibit the degranulation of mast cells for treatment of allergic disorders. The calcium levels in mast cells can regulate degranulation.25,26 The calcium level is significantly increased after mast cell activation.27 It is reported that after treatment with agents,28,29 which can decrease the intracellular calcium, mast cell degranulation is inhibited. In this study, AS showed a powerful inhibitory effect on the degranulation of RPMCs; however, it had no obvious cytotoxicity to the cell viability. AS effectively suppressed histamine release and deprived the influx of calcium into RPMCs. These results suggested that AS reduced IgE-mediated mast cell degranulation and histamine release through hindering the uptake of calcium into RPMCs.

Cytokines such as TNF-α, IL-1β, and IL-4 are essential in the progression to chronic allergic inflammation. TNF-α can promote inflammation, tissue fibrosis, and granuloma formation.30,31 IL-4, a major Th2 cytokine, is necessary for allergic responses, promoting a driving force of the generation of IgE in plasma B cells.32 IL-1β and IL-8 induce the adhesion molecule expression on the endothelium to induce inflammatory effector cells transmigration and activation.33,34 According to all the above reports, the inflammation and allergic symptoms may be suppressed through inhibiting the pro-inflammatory cytokine expression. In this study, we found that AS decreased the cytokine expression in the antigen-induced RBL-2H3 cells. NF-κB can simulate the cytokine expression in allergic inflammation.35−37 Inactivated NF-κB forms a trimer with IκBα in the cytosol. The activated NF-κB is transferred into the nucleus.38−40 Our results showed that AS up-regulated IκBα F

DOI: 10.1021/acs.jafc.7b01590 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Innovation of Jilin Youth Leader and Team (with Grant No. of 20140519013JH).

and inhibited the transfer of NF-κB into the nucleus stimulated by antigen-IgE cross-linking. Therefore, AS may inhibit proinflammatory cytokines through regulating the NF-κB pathway. Mast cell activation is dependent on Syk activation, which is regulated by the interaction between FcεRI and the factors of Lyn and Fyn.3 Lyn activates Syk and downstream signals.41−43 Syk plays a critical role in mast cell activation via activating downstream molecules, such as Gab2 and PLC-γ1, thus regulating inflammatory cytokine production.44 Therefore, the proteins of Lyn, Syk, Gab2, and PLC-γ1 were selected to test how AS suppressed mast cell activation. The results showed that AS suppressed the phosphorylation of Lyn, Syk, Gab2, and PLC-γ1 induced by antigens. It is reported that PLC/PKC are related to the MAP kinase activation and NF-κB activation.45,46 Moreover, the activated Gab2 increases the activation of the PI3K signaling pathway.45 The activation of these signaling pathways results in degranulation and expression of inflammatory cytokine genes in mast cells. Our data revealed that antigen-induced phosphorylation of Akt and MAPK was inhibited by AS. AS significantly suppressed FceεRI-mediated signaling transduction and thus attenuated the allergic response. Nrf2 can regulate glutathione S-transferase, NAD(P)H:quinine oxidoreductase-1 (NQO-1), HO-1, and γ-glutamylcysteine synthase, transcriptionally.47,48 HO-1 is a preferred target gene to prevent oxidative attack.49 Studies have shown that the inflammation could be affected through Nrf2/HO-1 pathways,50 Nrf2 signaling pathways negatively regulate the inflammatory responses mediated by NF-κB.51 Besides, there is a cross-talk between NF-κB and Nrf2 during the inflammation responses.52 In addition, Nrf2 is phosphorylated by several kinases, such as MAPKs, PI3K, and PKC, leading to its release from Keap1-mediated repression.53,54 Here, we found that AS activated Nrf2 and increased the HO-1 expression time dependently. The results indicate that Nrf2 and HO-1 might be used as the next attractive targets for research of AS pharmacological properties in mast cell-mediated allergic diseases. To sum up, we found that AS could inhibit FcεRI-mediated signaling activation, thus decreasing the degranulation and inflammation cytokine secretion in RBL-2H3 mast cells stimulated by antigens. Additionally, AS could reduce PCA, which is mediated by the mast cell in mice. These findings demonstrate that by suppressing NF-κB- and FcεRI-mediated signaling, AS has antiallergy activity in the mast cells.

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ABBREVIATIONS USED AS, asiaticoside; PCA, passive cutaneous anaphylaxis; RPMCs, rat peritoneal mast cells; TNF-α, tumor necrosis factor α; IL, interleukin; IgE, immunoglobulin E; PI3K, phosphatidylinositol 3-kinase; MAPKs, mitogen-activated protein kinase; NF-κB, nuclear factor-κB

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-433-2436152, Fax: +86-433-243-5136, E-mail: [email protected] and [email protected]. *Tel.: +86-433-243-5137, Fax: +86-433-243-5136, E-mail: [email protected] and [email protected]. ORCID

Guang Hai Yan: 0000-0001-8058-7822 Author Contributions ⊥

J.Z.J., J.Y., and G.Y.J. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study is sponsored by the National Natural Science Foundation of China (with Grant No. of 815600679, 81260016, and 81560004), and the Project of Research & G

DOI: 10.1021/acs.jafc.7b01590 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.jafc.7b01590 J. Agric. Food Chem. XXXX, XXX, XXX−XXX