Alisol B 23-Acetate Ameliorates Ovalbumin-Induced Allergic Asthma during Sensitization and Challenge Periods

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: Alisol B 23-acetate; Allergy; Asthma; Immunopharmacology; Spleen tyrosine kinase.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.