Oridonin Suppresses Mast Cell Degranulation and Alleviates Ovalbumin-Induced Allergic Rhinitis (2024)

Abstract

Incidence of type I allergies, such as hay fever, is continuously increasing in developed countries, including Japan. Type I allergies are triggered by chemical mediators, such as histamine, which are released via immunoglobulin E (IgE)-mediated mast cell degranulation. Therefore, medications inhibiting the synthesis, release, and receptor binding of these mediators are commonly used to manage type I allergy symptoms. As self-care disease prevention practices are gaining attention worldwide, regular consumption of food and supplements containing safe components inhibiting mast cell degranulation is a potential strategy to prevent allergic attacks. Here, we aimed to assess the ability of phytochemicals derived from edible plants to inhibit mast cell degranulation using the β-hexosaminidase release assay and investigate their cytotoxicity and efficiency in alleviating allergic symptoms. We found that oridonin, a diterpenoid isolated from Isodon japonicus Hara, strongly inhibited β-hexosaminidase release from both the RBL-2H3 rat cell line and mouse bone marrow-derived mast cells stimulated with dinitrophenyl (DNP)-conjugated human serum albumin after sensitization with DNP-IgE. Oridonin also inhibited β-hexosaminidase release induced by the calcium ionophore, A23187, in both cell types. Notably, oridonin did not adversely affect cell survival at concentrations necessary to inhibit β-hexosaminidase release. In a mouse model of ovalbumin (OVA)-induced allergic rhinitis, intraperitoneal administration of oridonin significantly reduced the nasal rubbing caused by intranasal OVA administration without affecting the serum levels of OVA-specific IgE. Therefore, oridonin could be an effective daily intake component to alleviate allergic diseases by inhibiting mast cell degranulation.

INTRODUCTION

Currently, many individuals are suffering from type I allergies, such as hay fever, necessitating the development of effective strategies to alleviate these allergic symptoms. The Coombs and Gell’s classification categorizes allergies into four types.¹⁾ Mast cells and antigen-specific immunoglobulin E (IgE) play key roles in the pathogenesis of type I allergies.^2,3) In type I allergies, exposure to an antigen (allergen) triggers the production of antigen-specific IgE, which binds to the high-affinity IgE receptor (FcεRI) on mast cells (sensitization). Upon subsequent exposure, the antigen binds to the antigen-specific IgE on mast cells (challenge). Activation of FcεRI initiates a signaling pathway, leading to increased intracellular Ca²⁺ levels in mast cells and release of granule contents, including chemical mediators, such as histamine (degranulation). These chemical mediators subsequently bind to receptors on surrounding cells, causing allergic reactions. Allergic diseases vary depending on the antigen and affected organ. For example, hay fever is a syndrome involving allergic rhinitis and conjunctivitis caused by pollen, which triggers allergic reactions in the nose and eyes. Type I allergies are conventionally treated with medications inhibiting the synthesis, release, and receptor binding of these chemical mediators.^4,5) However, many individuals do not use these medications due to concerns regarding their side effects and inconvenience of frequent medical visits.

Self-care practices to prevent diseases by managing daily lifestyle habits have been gaining attention worldwide. The practice of consuming food and herbal remedies to maintain health is common in many countries, including Japan. Therefore, identification of safe and allergy-suppressing components in food and herbal remedies and establishment of effective self-care strategies involving the intake of meals and supplements containing these components can aid in preventing type I allergies. Specifically, this approach can act as an effective countermeasure against type I allergies that occur unexpectedly owing to unpredictable antigen exposure. In this study, we investigated the mast cell degranulation-inhibiting ability of phytochemicals derived from edible plants used as food or herbal remedies. Previous studies have highlighted the beneficial effects of phytochemicals, including their antioxidant effects,⁶⁾ and suggested their self-care applications. Here, we identified oridonin as a potential self-care candidate and evaluated its safety and allergy-suppressing effects in mice and their cells.

MATERIALS AND METHODS

Reagents and Cell Line

A23187, chlorogenic acid, isoflavones, lutein, oridonin, quercetin, rosmarinic acid, and sulforaphane were purchased from Cayman Chemical Company (Ann Arbor, MI, U.S.A.). Capsaicin was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Lycopene was purchased from Selleck (Houston, TX, U.S.A.). Menthol, p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside, and 3,3′,5,5′-tetramethylbenzidine were purchased from Nacalai Tesque (Kyoto, Japan). Recombinant murine interleukin (IL)-3 was purchased from PeproTech (Rocky Hill, NJ, U.S.A.). Dinitrophenyl-conjugated human serum albumin (DNP-HSA), an anti-DNP IgE antibody (SPE-7), bovine serum albumin (BSA) solution, and ovalbumin (OVA) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Cytotoxicity lactate dehydrogenase (LDH) assay kit (WST) was purchased from Dojindo Laboratories (Kumamoto, Japan). Anti-mouse IgE antibody (LO-ME-2) was purchased from Bio-Rad (Hercules, CA, U.S.A.). Imject Alum and EZ-Link Sulfo-NHS-LC-Biotinylation Kit were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Anti-mouse c-kit (2B8) and anti-mouse FcεRIα (MAR-1) antibodies, and streptavidin-conjugated horseradish peroxidase were purchased from BioLegend (San Diego, CA, U.S.A.).

RBL-2H3, a rat basophilic leukemia cell line, was purchased from Riken (Ibaraki, Japan).

Mice

Female BALB/c mice (5-week-old) were purchased from Japan SLC Inc. (Shizuoka, Japan). During the experimental period, all mice were housed in standard cages in a temperature-controlled room under a 12-h light/dark cycle at the animal care facility of the Graduate School of Pharmaceutical Sciences, Osaka University. The mice were provided ad libitum access to standard laboratory mouse chow and drinking water. All animal experiments were approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences, Osaka University.

Cell Preparation

RBL-2H3 cells were maintained in the Eagle’s minimal essential medium (Nacalai Tesque) supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). Primary mast cells were generated from the bone marrow (BM) cells of BALB/c mice using IL-3, as previously described by Jensen et al.⁷⁾ After 6–9 weeks of culture, BM-derived mast cells (BMMCs) were used in subsequent experiments when the percentage of c-kit⁺ FcεRIα⁺ mast cells determined via flow cytometry was over 95%.

Cell Stimulation

Degranulation of RBL-2H3 cells and BMMCs was assessed by measuring the release of β-hexosaminidase, as described by Kuehn et al.,⁸⁾ with some modifications. RBL-2H3 cells were seeded at a density of 5 × 10⁴ cells/well in 96-well plates and sensitized overnight in a medium containing anti-DNP-IgE antibody (100 ng/mL). After replacing the medium with N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (10 mM HEPES [pH 7.4], 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na₂HPO₄-7H₂O, 5.6 mM glucose, 1.8 mM CaCl₂, 1.3 mM MgSO₄, and 0.04% BSA), the cells were pretreated with capsaicin (10 µM), chlorogenic acid (10 µM), isoflavone (10 µM), lutein (10 µM), lycopene (10 µM), menthol (10 µM), oridonin (2.5–10 µM), quercetin (2.5–10 µM), rosmarinic acid (10 µM), or sulforaphane (10 µM) for 30 min and stimulated with DNP-HSA (50 ng/mL) for 30 min. RBL-2H3 cells were prepared in the same manner without sensitization and stimulated with A23187 (10 µM) for 30 min after pretreatment with oridonin. BMMCs were sensitized with anti-DNP-IgE antibodies in the medium for differentiation. After washing and resuspending in HEPES buffer, BMMCs were treated under the same conditions as the RBL-2H3 cells. Culture supernatants were collected after centrifugation at 440 × g and 4 °C for 5 min and incubated with p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside in 40 mM citrate buffer (pH 4.5) at 37 °C for 1.5 h. After adding 0.4 M glycine buffer (pH 10.7), absorbance was measured at 405 nm with a reference filter of 620 nm using a GloMax^® Discover multimode plate reader (Promega, Madison, WI, U.S.A.). The percentage of β-hexosaminidase release was calculated using the following formula: β-hexosaminidase release (%) = (T − C)/(S − C) × 100, where C, T, and S indicate the absorbances of the supernatant when cells were not treated with test compounds, DNP-HSA, or A23187, when cells were treated DNP-HSA or A23187 after pretreatment with each test compound, and when cells were treated with DNP-HSA or A23187 alone, respectively.

Cell Survival Analysis

Survival of RBL-2H3 cells and BMMCs after treatment with oridonin (2.5–20 µM) for 2 h was measured using the cytotoxicity LDH assay kit (WST), according to the manufacturer’s instructions.

Induction of Allergic Rhinitis in Mice

All mice were immunized via intraperitoneal injection of 50 µg OVA and 1 mg Imject Alum in a total volume of 200 µL of phosphate-buffered saline (PBS) (Nacalai Tesque) on days 0, 7, and 14. The mice were divided into four groups (8 mice per group) as follows:

Group 1: Mice were intranasally administered 20 µL of PBS for seven consecutive days from days 21 to 27.

Group 2: Mice were intranasally challenged with 200 µg OVA dissolved in 20 µL PBS for seven consecutive days from days 21 to 27.

Groups 3 and 4: Mice were intraperitoneally injected with oridonin (10 mg/kg in Group 3 and 20 mg/kg in Group 4) 30 min before each intranasal OVA challenge from days 21 to 27.

After the final intranasal OVA challenge on day 27, the number of nasal rubs was recorded for 15 min. Blood samples were collected on day 28 and analyzed via enzyme-linked immunosorbent assay to measure the OVA-specific IgE titers in the serum.

Measurement of OVA-Specific IgE Antibody Levels in the Serum

Biotin-conjugated OVA was prepared using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit. Then, 96-well microtiter plates (Nunc-Immuno MicroWell 96-well Plates; Thermo Fisher Scientific) were coated with anti-mouse IgE antibody (5 µg/mL) in 100 mM bicarbonate buffer (pH 9.4) overnight at 4 °C and blocked with 3% BSA in PBS at room temperature for 1 h. Then, serum samples serially diluted with 1% BSA in PBS were added and incubated overnight at 4 °C. After incubation, biotin-conjugated OVA (1 : 1000) in 1% BSA in PBS was added and incubated at 37 °C for 1 h. Streptavidin-conjugated horseradish peroxidase (1 : 4000) diluted in PBS containing 1% BSA was added and incubated at 37 °C for 1 h. The wells were washed four times with PBS plus 0.05% Tween 20 between each step. After adding 3,3′,5,5′-tetramethylbenzidine, the plates were developed at room temperature. Finally, 1 M HCl was added, and absorbance of the solution was measured at 450 nm. Antibody titers are expressed as the reciprocal of the last dilution, yielding an extinction value higher than that of 1% BSA in PBS.

Statistical Analyses

Data were analyzed using the GraphPad Prism v.8.0 software (Boston, MA, U.S.A.). One-way ANOVA and Tukey–Kramer post-hoc tests were used for multi-group comparisons. Statistical significance was set at p < 0.05.

RESULTS

Oridonin Inhibits RBL-2H3 Cell Degranulation

To identify the candidate components inhibiting mast cell degranulation, we conducted a degranulation assay using the rat basophilic leukemia cell line, RBL-2H3. These cells degranulate in response to stimulation by antigen-IgE binding or A23187 calcium ionophore. Degranulation was assessed by measuring the release of β-hexosaminidase, a granular enzyme found in both mast cells and basophils. In this study, we investigated capsaicin, chlorogenic acid, isoflavone, lutein, lycopene, menthol, oridonin, rosmarinic acid, and sulforaphane as potential mast cell degranulation-inhibiting phytochemicals based on relevant information from previous reports suggesting that they may suppress mast cell activation or exert in vivo anti-allergic effects.^9–16) Quercetin, a mast cell degranulation inhibitor, was used as a positive control. DNP-IgE-sensitized RBL-2H3 cells were treated with 10 µM of each phytochemical and stimulated with DNP-HSA. Among the tested phytochemicals, oridonin, a diterpenoid isolated from Isodon japonicus Hara,¹⁷⁾ significantly inhibited IgE-mediated β-hexosaminidase release from RBL-2H3 cells (Fig. 1). Sulforaphane also inhibited β-hexosaminidase release from cells but to lesser extent than oridonin.

We investigated the concentration-dependent inhibitory effect of oridonin on RBL-2H3 cell degranulation. Oridonin inhibited IgE-mediated β-hexosaminidase release in a concentration-dependent manner, achieving nearly complete inhibition at 20 µM (Fig. 2A). Its inhibitory effect was comparable to that of quercetin. Additionally, oridonin inhibited β-hexosaminidase release induced by A23187 in a concentration-dependent manner (Fig. 2B). Notably, oridonin barely affected cell survival at the concentration range used to inhibit RBL-2H3 cell degranulation (Fig. 2C). These findings suggest oridonin as an effective candidate for the safe inhibition of mast cell degranulation.

Oridonin Strongly Inhibits Primary Mouse Mast Cell Degranulation

Next, we evaluated whether oridonin inhibits primary mast cell degranulation. Mouse BMMCs were treated with oridonin at a concentration range of 10–20 µM, which effectively inhibited RBL-2H3 cell degranulation. Pretreatment with oridonin significantly inhibited IgE-mediated β-hexosaminidase release from BMMCs (Fig. 3A). Inhibitory effect of oridonin was comparable to that of quercetin. Oridonin also strongly inhibited A23187-induced β-hexosaminidase release from BMMCs (Fig. 3B). Notably, oridonin did not affect BMMC survival at the tested concentration range (Fig. 3C). Therefore, oridonin inhibits primary mast cell degranulation without compromising the cell viability.

Oridonin Alleviates Allergic Rhinitis Symptoms in Mice

Mast cells play critical roles in allergic rhinitis, serving as potential therapeutic targets.^18,19) In this study, we evaluated the in vivo effects of oridonin using a mouse model of allergic rhinitis (Fig. 4A), which was characterized by symptoms dependent on the presence of mast cells.^20,21) After immunization with OVA, OVA in PBS was administered intranasally for seven consecutive days to induce rhinitis symptoms. Oridonin was intraperitoneally administered 30 min before intranasal OVA administration. On the final day, number of nasal rubs after OVA administration was counted. The number of nasal rubs was significantly increased in mice administered OVA in PBS compared to that in mice administered PBS alone (Fig. 4B). Oridonin treatment significantly reduced the number of nasal rubbing episodes in a dose-dependent manner. We also analyzed the effect of continuous oridonin administration on OVA-specific IgE production. Notably, oridonin did not affect the serum OVA-specific IgE titers (Fig. 4C). These findings suggest that oridonin alleviates allergic rhinitis symptoms without affecting OVA-specific IgE production.

DISCUSSION

Oridonin exerts various beneficial effects, including anti-cancer effects.²²⁾ To the best of our knowledge, this study is the first to demonstrate the mast cell degranulation-inhibiting ability of oridonin. Although the bioavailability of oridonin is relatively low,²³⁾ it exerts beneficial effects in animals fed a diet supplemented with oridonin.^24–26) Therefore, continuous intake of a sufficient dose of oridonin possibly inhibits cell degranulation in humans. This study also demonstrated the mast cell degranulation-inhibiting ability of sulforaphane for the first time. Therefore, intake of food containing beneficial phytochemicals with degranulation-inhibiting properties, such as oridonin and sulforaphane, is an effective approach to suppress allergies. Many ongoing studies are focusing on enhancing the bioavailability of oridonin.²⁷⁾ In the future, supplements with improved oridonin bioavailability can be used in self-care measures to prevent or alleviate allergic reactions.

In this study, oridonin inhibited the degranulation of mouse BMMCs induced by both IgE antigen and A23187, indicating that oridonin acts downstream of Ca²⁺ influx, leading to cell degranulation. Related to the finding that oridonin is less effective in inhibiting degranulation in RBL-2H3 cells than in BMMCs when stimulated with A23187, a previous study demonstrated that Ca²⁺ influx occurs more intensely in RBL-2H3 cells stimulated with A23187 than in BMMCs.²⁸⁾ However, it has been reported that upon stimulation by antigen-IgE binding, Ca²⁺ influx in RBL-2H3 cells and BMMCs occurs at comparable levels. Furthermore, Ca²⁺ influx in RBL-2H3 cells stimulated with A23187 is shown to be stronger than that induced by antigen-IgE binding. In this study as well, the downstream signals of Ca²⁺ influx might be activated to a greater extent in A23187-stimulated RBL-2H3 cells than BMMCs. Increase in intracellular Ca²⁺ levels and the subsequent activation of protein kinase C trigger the degranulation machinery.²⁹⁾ The granules undergo granule–granule fusion events mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors and translocate from the cytoplasm to the plasma membrane in a microtubule-dependent manner. Depolymerization of cortical actin regulated by coronin 1A and coronin 1B facilitates the fusion of granules with the plasma membrane, leading to degranulation. Oridonin does not inhibit protein kinase C.³⁰⁾ However, its effects on other regulatory proteins remain unknown, warranting further studies.

Ca²⁺ influx is critical for the degranulation of basophils and eosinophils, both of which express FcεRI-like mast cells and exacerbate allergic reactions. Consequently, oridonin may suppress allergic symptoms by broadly inhibiting the degranulation of granulocytes, including mast cells. Additionally, we previously demonstrated that oridonin inhibits particulate irritant-induced IL-1α release from macrophages.³¹⁾ When particulate irritants, such as PM2.5, are inhaled into the lungs, they stimulate alveolar macrophages to release IL-1α, which induces the production of antigen-specific IgE via the formation of inducible bronchus-associated lymphoid tissue.³²⁾ Therefore, oridonin may exert potent preventive effects against allergic respiratory diseases triggered by airborne particles through various mechanisms, including the suppression of mast cell degranulation and macrophage IL-1α release. Overall, our findings suggest that oridonin can be included in self-care strategies for allergy prevention. However, further studies are necessary to elucidate its action mechanisms and evaluate its therapeutic potential for conditions other than allergic rhinitis.

Acknowledgments

We would like to thank A. Sato for secretarial assistance, T. Namba and T. Masaoka for experimental support. This work was supported by the Undergraduate Student-Initiative Research Encouragement Project of Osaka University Foundation for the Future, Japan Society for the Promotion of Science KAKENHI (Grant numbers: 22H02766, 23K18421 to T.S., and 24K02687 to N.T.), Takeda Science Foundation (to T.S.), and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under Grant number: JP24ama121052.

Conflict of Interest

The authors declare no conflict of interest.

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