Biomolecules & Therapeutics 2023; 31(6): 611-618  https://doi.org/10.4062/biomolther.2023.032
Alisol B 23-Acetate Ameliorates Ovalbumin-Induced Allergic Asthma during Sensitization and Challenge Periods
Ki-Hyuk Nam1 and Dong-Soon Im1,2,*
1Department of Pharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul 02446,
2Department of Fundamental Pharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02446, Republic of Korea
*E-mail: imds@khu.ac.kr
Tel: +82-2-961-9377, Fax: +82-2-961-9580
Received: February 18, 2023; Revised: April 15, 2023; Accepted: April 17, 2023; Published online: June 15, 2023.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Rhizome of Alisma orientale has been used as a traditional medicine for treating kidney diseases in East Asian countries. Its inhibitory effects on hypersensitivity responses have been reported for methanol extracts, with alisol B 23-acetate (AB23Ac) being the most active constituent among six terpenes in inhibiting the direct passive Arthus reaction. However, whether AB23Ac has efficacy against allergic asthma has not been tested to date. The in vivo efficacy of AB23Ac in an ovalbumin (OVA)-induced allergic asthma mouse model was evaluated by administrating AB23Ac before OVA sensitization or OVA challenge in BALB/c mice. AB23Ac suppressed antigen-induced degranulation of RBL-2H3 mast cells in a concentration-dependent manner. The administration of AB23Ac both before OVA sensitization and OVA challenge greatly lowered pulmonary resistance and the increase in immune cell counts and inflammatory responses around the peribronchial and perivascular regions. In addition, the inflammatory cytokine levels of Th1/Th2/Th17 cells in the bronchoalveolar lavage fluid decreased in the AB23Ac-treated groups. AB23Ac reduced the number of PAS-stained cells in the lungs. Furthermore, a computer modeling study indicated that AB23Ac can bind tightly to spleen tyrosine kinase (Syk). These results suggest that AB23Ac may ameliorate allergic asthma by suppressing immune responses in dendritic cells during sensitization and in mast cells during challenge periods.
Keywords: Asthma, Allergy, Alisol B 23-acetate, Immunopharmacology, Spleen tyrosine kinase
INTRODUCTION

Asthma is a chronic inflammatory disease of the lungs that results in increased airway hyper-responsiveness (AHR), mucus hyper-production, and bronchial wall remodeling (Scarlata and Incalzi, 2022). Bronchial asthma is prevalent worldwide, especially in young children, and is a life-threatening respiratory condition in developed countries (Bhatiaa et al., 2022). In allergic asthma, the immune response occurs in two steps: an initial exposure to sensitizing antigens and subsequent repeated exposures to antigens (Hesse et al., 2022). Bronchial dendritic cells internalize and present antigens to naïve T cells in the mediastinal lymph nodes during antigen sensitization (Hesse et al., 2022). IgE-coated mast cells and eosinophils drive immune responses in the airways during antigen exposure (Hesse et al., 2022). Inhaled glucocorticosteroids, long-acting β2 agonists, and oral leukotriene D4 antagonists are vital for treating asthma symptoms (Duman et al., 2022). However, alternative therapeutic approaches are required to overcome the frequently occurring steroid-induced side effects of long-time use.

Rhizome of Alisma orientale has been used in traditional decoctions for kidney diseases in East Asian countries. For examples, rhizome of Alisma orientale has been included in Palmijihwang-hwan, Choreito, and Jin Gui Shen Qi Wan in Korea, Japan, and China, respectively (Kubo et al., 1997; Kao et al., 2018; Sung et al., 2019). An initial study of its anti-nephritis effect was conducted to determine the anti-hypersensitivity actions of six terpene components in its methanol extracts (Kubo et al., 1997). The methanol extract also inhibited zymosan-induced hind paw edema in rats (Matsuda et al., 1998) and its ethanol extract significantly inhibited antigen-induced β-hexosaminidase release by RBL-2H3 cells (Lee et al., 2012). Among the isolated components, alisol B 23-acetate (AB23Ac) was found to be the most active terpene in terms of inhibition of the direct passive Arthus reaction (Kubo et al., 1997). AB23Ac also inhibited complement-induced hemolysis via the classical pathway (Matsuda et al., 1998). AB23Ac ameliorates delayed-type hypersensitivity responses in mice (Lee et al., 2012). AB23Ac inhibited the IgE-mediated degranulation of bone marrow-derived mast cells (Shao et al., 2018), and it also attenuated IgE-mediated passive cutaneous anaphylaxis (Shao et al., 2018). However, the effects of AB23Ac on allergic asthma have not yet been evaluated in vivo. In the present study, we tested whether AB23Ac suppresses ovalbumin (OVA)-induced allergic asthma by administrating it before OVA sensitization or challenge in BALB/c mice.

MATERIALS AND METHODS

Materials

AB23Ac was purchased from APExBIO (Cat no. N1550, Houston, TX, USA). OVA and alum were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

Rat RBL-2H3 mast cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured at 37°C in 5% CO2 in a humidified incubator and maintained in Dulbecco’s modified Eagle medium (DMEM)-high glucose containing 10% (v/v) heat-inactivated fetal bovine serum along with 2 mM glutamine, 100 U/mL penicillin, 1 mM sodium pyruvate, and 50 μg/mL streptomycin (Lee et al., 2023).

Animals

Daehan Biolink Co. Ltd. (Seoul, Korea) provided five-week-old female BALB/c mice. The mice were housed in a laboratory animal facility at Kyung Hee University (Seoul, Korea) and provided water and food ad libitum. The Institutional Animal Care Committee of the university reviewed and approved the study protocol (Approval Number, KHSASP-23-013).

Assessment of degranulation

Degranulation of RBL-2H3 cells was assessed by measuring the β-hexosaminidase activity in the medium. Mouse monoclonal anti-dinitrophenyl immunoglobulin E (DNP-IgE) and human DNP albumin were used to induce degranulation (Lee and Im, 2021b).

Asthma induction in mice and AB23Ac administration

Six-week-old female BALB/c mice were divided into four treatment groups (n=5): a PBS-injected control group, an OVA-injected asthma group, a group treated with AB23Ac (60 mg/kg) before sensitization plus OVA-injection, and a group treated with AB23Ac before challenge plus OVA-injection. Sensitization was by intraperitoneal injection of 50 μg OVA and 1 mg aluminum hydroxide on the day 0 (D0) and D14 (sensitization). OVA exposure was by delivery of nebulized 1% OVA or PBS for 30 min using an ultrasonic nebulizer (Philips) on D28, D29, and D30 (challenge). AB23Ac was administered via intraperitoneal injection 30 min before OVA sensitization (D0 and D14) or 30 min before OVA challenge (D28, D29, and D30). Bronchoalveolar lavage fluid (BALF) was collected from lungs on D32, and the population of BALF cells was analyzed after staining (Lee and Im, 2021a).

BALF cell counting and analysis

The immune cells in the BALF were collected in 0.5 mL PBS. Then, 25 μL of this sample was mixed with 25 μL of Wright’s solution, and the mixed 50 μL was loaded into the hemocytometer. After counting the cell numbers, the total cell numbers were calculated by multiplication. For the May-Grünwald Giemsa staining, BALF cells in 50 μL of cell suspension were adhered to a glass slide using Cellspin® centrifuge (Hanil Electric, Seoul, Korea), fixed in MeOH for 30 s, and then were stained with May-Grünwald solution for 8 min, followed by Giemsa solution for 12 min. Based on cellular staining and morphology characteristics, cells were classified as eosinophils, lymphocytes, and macrophages. In each case, 200 cells were counted and the percentage of each cell type was calculated.

Measuring AHR to methacholine

The day after the final OVA challenge, AHR was evaluated using a non-invasive lung function measurement Model PLY-UNR-MS2 (EMKA Technologies, Paris, France). After placing the mice in a barometric plethysmographic chamber (Model PLY-UNR-MS2; EMKA Technologies), the baseline was recorded for 3 min and the enhanced pause (Penh) was calculated according to the manufacture’s protocol. The results are expressed as the percentage increase in Penh following challenge with increasing concentrations of methacholine (0, 6.25, 12.5, 25 and 50 mg/mL) (Lee and Im, 2022).

Histological examination of the lung

Lung tissue sections prepared from the lungs were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) to evaluate immune cell infiltration and mucus-producing cells, respectively (Lee and Im, 2022).

A treatment-blind observer measured the degree of lung inflammation on a subjective scale of 0-3. PAS-stained mucin-secreting cells around the bronchioles were counted in two lung sections per mouse. Mucous production was expressed as the number of PAS-positive cells per millimeter of bronchiole after measuring the length of the bronchial basal lamina using ImageJ software (National Institute of Health, Bethesda, MD, USA) (Lee and Im, 2022).

Measurement of the levels of total serum IgE and IL-13

Serum IgE and bronchoalveolar lavage fluid (BALF) IL-13 levels in the mice were evaluated using ELISA kits (eBioscience, San Diego, CA, USA). Capture and biotinylated detection antibodies specific for IL-13 were obtained from eBioscience (Cat no. 14-7043-68 and 33-7135-68B). The absorbance was measured at 450 nm (Son et al., 2022).

Molecular docking

To investigate the exact mechanism by which AB23Ac activates Syk, we performed molecular docking in silico with Autdock 4.2 (The Scripps Research Institute, La Jolla, CA, USA). The crystal structure of human SYK was used as the receptor template (PDB code:6VOV). To determine the ligand conformer with the lowest binding energy, AB23A structures were built three dimensionally and optimized using ChemDraw Ultra 11 (CambridgeSoft Corporation, Cambridge, MA, USA). Chimera 1.10 (UCSF Chimera, San Francisco, CA, USA) (Kim et al., 2021) was used to create the figures.

Statistics

The statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to compare the differences among multiple groups. Data are expressed as means ± standard error of the mean (SEM). Differences were considered statistically significant at p<0.05.

RESULTS

AB23Ac suppressed degranulation of mast cells

Lung mast cells respond to antigens during asthma attacks. Multivalent antigen exposure crosslinks the IgE-FcεRI complex in the mast cell membrane, turning on intracellular activation of tyrosine protein kinases such as Syk (also known as spleen tyrosine kinase), Lyn, and Fyn, ultimately leading to degranulation of vesicles (Shao et al., 2018). Degranulation of mast cells results in the release of many early-step mediators of allergic asthma, including histamine, chemokines, cytokines, neutral proteases (chymase and tryptase), and leukotrienes. Mast cell degranulation was evaluated as increased β-hexosaminidase activity in the medium after exposure of RBL-2H3 rat basophilic leukemia cells to human serum albumin (antigen) (Fig. 1). Treatment of AB23Ac inhibited β-hexosaminidase release in a concentration-dependent manner (Fig. 1). The AB23Ac-induced inhibition of β-hexosaminidase release was significant at concentrations of 5, 10, and 20 μM (Fig. 1), which is consistent with a previous report that AB23Ac inhibited IgE-mediated degranulation of bone marrow-derived mast cells (Lee et al., 2012; Shao et al., 2018).

Figure 1. AB23Ac reduces antigen-induced degranulation in RBL-2H3 mast cells. After sensitization with anti- dinitrophenyl immunoglobulin E (DNP-IgE) for 18 h, RBL-2H3 cells were challenged with dinitrophenyl-human serum albumin (DNP-HSA). AB23Ac treatment was performed at the indicated concentrations 30 min before antigen challenge. Basal degranulation shows samples without IgE and HSA, and the positive control of antigen-induced degranulation shows samples with IgE and HSA. The results are presented as means ± the standard error (SE) of three independent experiments. ***p<0.001 vs. the HSA-untreated group. #p<0.05, ##p<0.01, ###p<0.001 vs. the HSA-treated group.

Effects of AB23Ac on the AHR and serum IgE titers during OVA-induced asthma

The in vitro inhibitory effect of AB23Ac on mast cell degranulation was further investigated in an OVA-induced allergic asthma model of BALB/c mice in vivo. AB23Ac (60 mg/kg) was injected intraperitoneally 30 min before OVA sensitization or challenge. Penh value was measured to determine the effects of AB23Ac on AHR. OVA-sensitized mice exhibited significant increases in Penh values compared to those of control mice at dosages from 6.25 to 50 mg/mL methacholine, indicating that the asthma induction was successful. Both AB23Ac treatments significantly reduced the increased Penh values in OVA-induced mice at dosages from 6.25 to 50 mg/mL methacholine to approximately the control level (Fig. 2A). The serum IgE concentration was significantly suppressed in the OVA group compared to that in the PBS group, implying that asthma induction was successful. However, the OVA-induced increase in serum IgE concentrations were significantly suppressed in the OVA+AB23Ac group compared to that in the OVA group (Fig. 2B). In particular, it was significant in the OVA+AB23Ac treatment before antigen sensitization, but was not statistically significant in the OVA+AB23Ac treatment before antigen challenge (Fig. 2B).

Figure 2. Effects of AB23Ac treatment on airway hyper-responsiveness to methacholine in an OVA-induced murine asthma model and IgE levels in serum. (A) Airway hyper-responsiveness was measured as Penh (enhanced pause) in mice treated with AB23Ac (60 mg/kg) or PBS after challenge with increasing concentrations of methacholine. PBS: PBS-treated mice, OVA: OVA-challenged mice, OVA+AB23Ac sensitization: OVA-challenged mice treated AB23Ac before sensitization, OVA+AB23Ac challenge: OVA-challenged mice treated AB23Ac before challenge. (B) ELISA was used to measure the protein levels of IgE in serum. The results are presented as means ± the standard error of the mean (SEM) (n=5). **p<0.01, ***p<0.001 vs. the PBS-treated group, ##p<0.01, ###p<0.001 vs. the OVA-treated group.

AB23Ac suppressed the increase in eosinophil and lymphocyte counts in the BALF

Immune cells infiltrated and increased significantly in the BALF of the OVA group compared to that of the PBS group, indicating successful asthma induction (Fig. 3A). AB23Ac treatment before antigen sensitization or challenge significantly suppressed the increase in the total cell number (Fig. 3B). The eosinophil and lymphocyte counts were significantly increased by OVA and decreased by both AB23Ac treatments, whereas the macrophage count was not significantly changed after OVA or AB23Ac treatment (Fig. 3B, 3C).

Figure 3. AB23Ac inhibits OVA-induced immune cell accumulation in BALF. (A) Mice were sensitized with OVA twice by i.p. injection on D0 and D14 and were subsequently challenged on D28, D29, and D30 with nebulized OVA. AB23Ac was administrated intraperitoneally at a dose of 60 mg/kg 30 min before the OVA sensitization or before the OVA challenge. The cells in the BALF were stained using May-Grünwald stain and counted. (B) Total cell counts, eosinophils, and macrophages in the BALF. (C) Lymphocyte counts in BALF. The results are presented as means ± the SEM cell count values (n=5). *p<0.05, **p<0.01, ***p<0.001 vs. the PBS-treated group, #p<0.05, ##p<0.01 vs. the OVA-treated group.

AB23Ac suppressed morphologic changes and inflammation in the lungs

H&E and PAS staining were conducted on lung samples to determine the effects of AB23Ac on histological changes in asthma. H&E-stained sections from mice exposed to OVA revealed airway inflammation in lung tissues (Fig. 4A). Eosinophils were observed as small, dark blue dots in H&E staining of lung sections (Fig. 4A). Few eosinophils were found in the PBS control group, whereas many eosinophils were detected around the peribronchial regions in the OVA group (Fig. 4A). Conversely, airway inflammation was reduced in lung tissue sections from mice treated with AB23Ac before OVA sensitization or challenge (Fig. 4A).

Figure 4. AB23Ac protects against airway inflammation and mucin production. (A) H&E-stained sections of lung tissues from the PBS, OVA, and AB23Ac (60 mg/kg)-treated OVA groups. The small navy blue dots around the bronchioles are eosinophils. Eosinophils were rarely observed in the PBS group, whereas they accumulated extensively around the bronchioles in the OVA group (green arrows). (B) Periodic acid-Schiff (PAS)/hematoxylin-stained sections of lung tissues from the PBS, OVA, and AB23Ac (60 mg/kg)-treated OVA groups. In PAS staining, mucin is stained purple. In the OVA group, a darker and thicker purple color was observed surrounding the bronchioles compared with that in the PBS group (red arrows). However, the eosinophil accumulation was less pronounced in the OVA+AB23Ac group than in the OVA group. (C) The lung inflammation was semi-quantitatively evaluated; histological findings were scored as described in the Materials and Methods section. (D) Mucous production was evaluated by counting the number of PAS-positive cells (red arrows) per mm of bronchioles (n=5 per group). Values represent means ± the SEM (n=5). ***p<0.001 vs. the PBS-treated group, ##p<0.01, #p<0.05 vs. the OVA-treated group.

Histological studies using PAS staining of lung samples revealed an increase in mucin hypersecretion. PAS-positive goblet cells were detected around the bronchial airways in the OVA and OVA plus AB23Ac groups (Fig. 4B), indicating increased mucin production and hyperplasia of goblet cells. Dark violet spots surrounded the bronchioles in the OVA group (Fig. 4B). AB23Ac treatment significantly decreased the number of PAS-positive goblet cells around the bronchial airway (Fig. 4B).

Lung inflammation was assessed using a subjective scale of 0-3. The average inflammation score in the OVA-treated group was approximately 2.0, which was significantly reduced after AB23Ac treatment before sensitization or challenge (Fig. 4C). Mucin production was semi-quantitatively analyzed by measuring the number of PAS-positive cells in the bronchioles (Fig. 4D). PAS-stained cells were rare in the PBS-treated group. However, there were approximately 100 PAS-positive cells/mm in the OVA-treated group, and the numbers were significantly reduced after AB23Ac treatment before sensitization or challenge (Fig. 4D).

AB23Ac inhibited asthma-induced cytokine expression in the BALF

IL-4 and IL-13 are type 2 proinflammatory cytokines and are central in the pathogenesis of allergic asthma, particularly the synthesis of IgE, eosinophil accumulation in the airway wall, and hypersecretion of mucus in epithelial cells (Akdis et al., 2020). In addition, the late-stage pathogenesis of asthma progresses in combination with Th1 and Th17 cytokines. Therefore, changes in the mRNA levels of the Th2 cytokines IL-4 and IL-13, the Th1 cytokine IFN-γ, and the Th17 cytokine IL-17A were measured by qPCR in BALF cells. Fig. 5 shows elevated mRNA levels of all four cytokines in BALF immune cells from the OVA group. These increases were inhibited by AB23Ac in the OVA plus AB23Ac groups (Fig. 5).

Figure 5. AB23Ac treatment inhibits the mRNA expression of cytokines in BALF cells. Analysis of the mRNA expression of the Th2 cytokines IL-4 and IL-13, the Th1 cytokine IFN-γ, and the Th17 cytokine IL-17A in the BALF cells. (A) IL-4, (B) IL-13, (C) IFN-γ, and (D) IL-17A. The relative mRNA levels of the cytokines were quantified relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The values are represented as means ± the SEM (n=5). *p<0.05, **p<0.01 vs. the PBS-treated group, #p<0.05, ##p<0.01 vs. the OVA-treated group.

Similarly, changes in the mRNA levels of IL-4, IL-5, IL-13, IL-17A, and IL-33 in the lungs were measured by qPCR. Fig. 6 shows elevated mRNA levels of these five cytokines in the lungs of the OVA group, and AB23Ac treatment both before OVA challenge and sensitization significantly reduced the OVA-induced increase (Fig. 6).

Figure 6. AB23Ac treatment inhibits the mRNA expression of cytokines in the lungs. Analysis of mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13, the Th17 cytokine IL-17A, and IL-33 in the lung tissues. (A) IL-4, (B) IL-5, (C) IL-13, (D) IL-17A, and (E) IL-33. The relative mRNA levels of the cytokines were quantified relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The values are represented as means ± the SEM (n=5). **p<0.01, ***p<0.001 vs. the PBS-treated group, #p<0.05, ##p<0.01, ###p<0.001 vs. the OVA-treated group.

AB23Ac suppressed OVA-induced increase in BALF IL-13 levels

Th2 cytokines such as IL-13 play an essential role in the development of allergic asthma by inducing eosinophil recruitment, goblet cell metaplasia, and mucus hypersecretion. The protein level of the Th2 cytokine IL-13 in the BALF was assessed by ELISA. IL-13 levels were elevated in the OVA-induced group compared to those in the PBS-treated control group, and the elevation in IL-13 levels was significantly reduced by both AB23Ac treatments (Fig. 7).

Figure 7. Effect of AB23Ac on IL-13 levels in BALF. ELISA was used to measure the protein levels of IL-13 in the BALF. The results are presented as means ± the standard error of the mean (SEM) (n=5). *p<0.05 vs. the PBS-treated group, #p<0.05 vs. the OVA-treated group.

Docking modeling of AB23Ac with Syk

AB23Ac has been reported to inhibit the phosphorylation of Syk, thereby blocking its activation and downstream signaling pathways (Shao et al., 2018). Therefore, we analyzed the interaction between AB23Ac and Syk using the computational modeling program Autodock 4.2 (Kim et al., 2021). AB23Ac binds in the active site pocket, which is normally occupied by ATP in Syk with a minimum energy of –9.21 kcal/mol (Fig. 8A). AB23Ac formed hydrogen bonds with Lys375, Leu377, Lys458, and Asp512, and hydrophobic interactions with surrounding residues, such as Val385, Lys402, Val433, Met448, Leu501, and Ser511 (Fig. 8B). The interaction between AB23Ac and Lys375, Leu377, Lys458, Asp512 occurred with bonding distances of 1.776 Å, 2.085 Å, 3.858 Å, and 3.250 Å, respectively (Fig. 8B).

Figure 8. Molecular docking model of AB23Ac binding to Syk. (A) Schematic representation of the ternary complex of AB23Ac (lime green) with Syk (PDB Code: 6VOV) visualized using Chimera 1.10 (UCSF Chimera). (B) Binding model of AB23Ac in the Syk ATP-binding pocket. The amino acid residues for hydrogen bonding are shown in red and hydrophobic residues are in cyan.
DISCUSSION

Rhizome of Alisma orientale has traditionally been used to treat kidney diseases in East Asian countries (Kubo et al., 1997; Kao et al., 2018; Sung et al., 2019). The initial question was whether rhizome of Alisma orientale is active against nephritis and which components contribute to its anti-nephritic effect (Kubo et al., 1997). Methanol and ethanol extracts and terpenes have been shown to inhibit hypersensitivity responses (Matsuda et al., 1998; Lee et al., 2012). Among these components, AB23Ac results in the greatest level of immune suppression (Kubo et al., 1997; Matsuda et al., 1998; Lee et al., 2012). While AB23Ac inhibits IgE-mediated degranulation of mast cells and attenuates IgE-mediated passive cutaneous anaphylaxis, inhibition of Syk phosphorylation has been suggested as a mode of action of AB23Ac (Shao et al., 2018). In this study, we demonstrated inhibition of antigen-induced degranulation of mast cells by AB23Ac in vitro, amelioration of OVA-induced allergic asthma in vivo, and a molecular binding model of the interaction of AB23Ac with Syk in silico.

The present observation of the inhibitory effect of AB23Ac on antigen-induced degranulation confirms previous studies (Lee et al., 2012; Shao et al., 2018). This inhibition strongly supports the anti-asthmatic effect of AB23Ac treatment before challenge because mast cell degranulation is the key step during antigen challenge. This inhibitory effect on mast cells may contribute to and support the previous in vivo anti-immune responses of AB23Ac in IgE-mediated passive cutaneous anaphylaxis and hapten-induced dermatitis symptoms in NC/Nga mice (Lee et al., 2012; Shao et al., 2018).

However, we observed a strong inhibitory effect of AB23Ac before antigen sensitization, implying important action of AB23Ac during antigen sensitization, such as functional inhibition of dendritic cells. In addition, the inhibitory effects of AB23Ac treatment before antigen sensitization were statistically significant and superior in terms of serum IgE and BALF IL-13 levels than the effects of AB23Ac treatment before antigen challenge, thus implying that AB23Ac inhibits the initial step(s) in dendritic cells during antigen sensitization. A previous in vitro study reported that AB23Ac treatment reduced the expression of CD80, MHC II, and CD86 in cholesterol-loaded dendritic cells and reduced CD4+ T-cells activation and inflammatory IL-12 and IFN-γ production (Sun et al., 2021), supporting the inhibitory effects of AB23Ac during antigen sensitization.

To date, two molecular targets of AB23Ac have been suggested: farnesoid X receptor (FXR), a nuclear receptor, and Syk. First, AB23Ac has been proposed to be an agonist of FXR (Meng et al., 2014), and its activation results in increased expression of genes that are important for hepatocyte proliferation and promotes liver regeneration (Meng et al., 2014). A molecular docking study greatly elucidated the binding of AB23Ac to FXR (Meng et al., 2014). However, although Syk has been suggested to be a target of AB23Ac, no modeling studies have been published to date (Shao et al., 2018). Because Syk is the initial signaling molecule of IgE-FcεRI complexes in mast cells, we found a very low energy (–9.21 kcal/mol) for the binding of AB23Ac with Syk using a computational docking program. The suggested amino acid residues for hydrogen bonding and hydrophobic interactions and the calculated bonding distances strongly support Syk as a molecular target of AB23Ac in anti-allergic asthma.

Allergen-induced allergic responses in the lung are initiated, when the resident dendritic cells in the airway epithelium perceive allergens as harmful and present the antigens to naïve T cells in the draining lymph nodes. Antigen-specific T cells are induced by differentiation of naïve T cells into Th2 effector cells in the allergic responses. Dendritic cells express Syk proteins, but T cells do not (Matsubara et al., 2006a). Therefore, significant inhibition of OVA-induced AHR and inflammatory responses by AB23Ac treatment before OVA sensitization implies that suppression of Syk activity in dendritic cells was effective in this group. Previously, Syk was found to be indispensable not only for the maturation of dendritic cells but also for antigen presentation to T cells (Sedlik et al., 2003; Nakashima et al., 2004). Phosphorylated Syk induced ERK1/2 phosphorylation and transcription factor NF-κB activation in dendritic cells (Sedlik et al., 2003). The importance of Syk activity in degranulation has also been previously reported in human cultured mast cells and murine bone marrow-derived mast cells (Shichijo et al., 2003; Matsubara et al., 2006b). A Syk inhibitor, R406, inhibited OVA-induced degranulation of passively IgE-sensitized murine mast cells (Matsubara et al., 2006b). Syk activation induced the phosphorylation of ERK1/2, JNK1/2, and ERK5 in murine mast cells (Matsubara et al., 2006b). Recently, Shao et al. (2018) confirmed three downstream signaling cascades of Syk in mast cells. Syk inhibition with a specific Syk inhibitor, Bay 61-3606, or AB23Ac 1) suppressed IgE-induced phosphorylation of PLCγ, intracellular Ca2+ increase, and degranulation, 2) attenuated phosphorylation of Akt and IKKα/β, degradation of Iκ-B, and translocation of NF-κB p65 to the nucleus, and 3) inhibited the phosphorylation of p38 MAPK, ERK, and cPLA2 (Shao et al., 2018). The last two signaling cascades originated from antigen/FcεRI-mediated Syk activation result in transcriptional upregulation of various inflammatory genes such as IL-6, TNF-α, and COX-2 (Shao et al., 2018). Although the phosphorylation of Syk, ERK, p38 MAPK, PLCγ, Akt, IκBα, and translocation of NF-κB have not been investigated in the present study, we presume that the administration of AB23Ac in vivo resulted in attenuated phosphorylation of Syk, ERK, p38 MAPK, PLCγ, Akt, IκBα, and translocation of NF-κB in the lung tissues based on the previous studies in human mast cell HMC-1, bone marrow-derived mast cells, RBL-2H3 cells, and dendritic cells (Sedlik et al., 2003; Shao et al., 2018). In vivo administration of R406 significantly inhibited the increases in levels of IL-13, which is consistent with our results (Matsubara et al., 2006a). However, R406 treatment did not induce any changes in the levels of IgEs, which is contrasting to our results (Matsubara et al., 2006a). This might imply that AB23Ac inhibit additional signaling molecule in IgE-producing B cells.

In summary, the present study of AB23Ac in inhibition of degranulation and amelioration of allergic asthma, as well as the binding model involving Syk strongly suggest that AB23Ac could be a possible candidate for the development of an anti-allergy and anti-asthma therapeutic medicines.

ACKNOWLEDGMENTS

This research was supported by the Basic Research Laboratory Program (BRL) and the Basic Science Research Program of the Korean National Research Foundation funded by the Korean Ministry of Science, ICT, and Future Planning (NRF-2020R1A4A1016142, NRF-2023R1A2C2002380, and NRF-2019R1A2C1005523).

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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