Degranulation In RBL-2H3 Cells Research Articles

Traditional cooking methods decreased the allergenicity of egg proteins.

Egg allergy is one of the most common food allergies globally. This study aimed to assess the impact of four traditional cooking methods on the allergenicity of egg proteins using a comprehensive strategy, including simulated gastrointestinal digestion in vitro, serology experiments, a rat basophilic leukemia (RBL)-2H3 cell degranulation model, and a passive cutaneous anaphylaxis (PCA) mice model, and the structure changes were detected by circular dichroism (CD)spectra and ultraviolet (UV) spectra. The results showed that the processed egg proteins were more readily digested compared to raw egg proteins. The serological experiments revealed a significant reduction in immunoglobulin E binding of egg proteins after thermal treatments (p<0.05), particularly after frying. Subsequently, the RBL-2H3 cell degranulation experiment demonstrated a marked decrease in the level of egg allergens-induced β-hexosaminidase release after cooking (p<0.05). Moreover, the results from the PCA mice model indicated that the increase in vascular permeability was effectively relieved in the treated groups, especially in frying group (p<0.05). Additionally, the α-helix and β-turn contents of processed egg proteins were significantly decreased (p<0.05) compared with native egg proteins. The UV spectra findings showed that all cooking treatments caused significant alterations in the tertiary structure, and fluorescence analysis indicated that cooking decreased the surface hydrophobicity of egg proteins. In conclusion, four traditional cooking methods reduced the allergenicity of egg proteins, particularly frying, and this reduction was associated with structural changes that could contribute to the destruction or masking of epitopes of egg allergens. PRACTICAL APPLICATION: Egg allergy has a serious impact on public health, and there is no ideal treatment method at present. This study demonstrated that four traditional cooking methods (boiling, steaming, baking, and frying) reduced the allergenicity of egg proteins, especially frying, and the results will provide a basis for the development of hypoallergenic egg products.

Degranulation of RBL-2H3 rat basophilic leukemia cells is synergistically inhibited by combined treatment with nobiletin and lactoferrin.

The aim of this study was to elucidate the anti-allergic effects of polymethoxyflavonoids in combination with milk proteins and the mechanism of inhibition. Three polymethoxyflavonoids and two milk proteins were exposed to the rat basophilic leukemia cell line RBL-2H3. β-hexosaminidase was used as an indicator of degranulation inhibition. The mechanism of inhibition was examined by measuring intracellular Ca2+ levels and western blot method. In the degranulation inhibition test with polymethoxyflavonoids and milk proteins alone, nobiletin was the strongest inhibitor in the polymethoxyflavonoid group and lactoferrin in the milk protein group. Next, co-stimulation with nobiletin and lactoferrin showed stronger synergistic degranulation inhibition than treatment with nobiletin or lactoferrin alone. Western blot analysis showed that co-stimulation with nobiletin and lactoferrin significantly downregulated the induction of phospholipase Cγ 1 phosphorylation. The degranulation response in RBL-2H3 cells was synergistically suppressed by co-stimulation of nobiletin and lactoferrin acting on both Ca2+-dependent and Ca2+-independent pathways.

Paedoksan ameliorates allergic disease through inhibition of the phosphorylation of STAT6 in DNCB-induced atopic dermatitis like mice

Various allergic diseases such as atopic dermatitis (AD), allergic rhinitis, and asthma are considered incurable conditions that have yet to be fully conquered. Paedoksan (PDS), an herbal preparation consisting of 14 medicines, displays effective anti-inflammatory and anti-allergic properties, yet its underlying molecular mechanism is unknown. This study aims to uncover PDS’s mechanism for treating allergic diseases and suggest its therapeutic potential. Through a network pharmacological prediction, its impact on signal transducer and activator of transcription 6 (STAT6) regulation, a sub-mechanism of interleukin 4 (IL-4), a major inflammatory cytokine involved in degranulation and allergy, was investigated in RBL-2H3 cells and an atopic mouse model. PDS inhibits immunoglobulin E (IgE)-induced degranulation and STAT6 phosphorylation evoked by IL-4 in granulocytes. The downregulation of phospho-STAT6 and thymic stromal lymphopoietin (TSLP) by PDS was confirmed in 2,4-dinitrochlorobenzene (DNCB)-induced mouse skin. The results demonstrate that PDS exhibited remarkable effects on degranulation and STAT6 phosphorylation in RBL-2H3 cells, as well as in an atopic mouse model. Furthermore, the main active components from PDS based on chromatographic analysis showed good accordance with PDS’s effects on RBL-2H3 cells. In summary, these findings collectively suggest that PDS holds the potential to effectively suppress inflammatory and allergic reactions by obstructing the target IL-4 protein and its downstream effects, as elucidated through a network pharmacological analysis.

Effects and potential mechanisms of Saposhnikovia divaricata (Turcz.) Schischk. On type I allergy and pseudoallergic reactions in vitro and in vivo

Ethnopharmacological relevanceThe incidence of allergic disease is constantly increasing, but its pathogenesis is not fully understood. Saposhnikovia divaricata (SD), called ‘Fangfeng’ in China, not only can be used for antipyretic, analgesic and anti-inflammatory as a traditional Chinese medicine, but also as an active ingredient in about 8% prescriptions. However, its effects on type I allergy and pseudoallergy have not been clarified. Aim of the studyTo explore the treatment and potential mechanisms of SD and its major bioactive component Prim-O-glucosylcimifugin (POG) on type I allergy and pseudoallergy in vitro and in vivo. Materials and methodsThe inhibitory effect of SD decoction and POG on type I allergy and its possible mechanism were evaluated by using RBL-2H3 cells model in vitro and the passive cutaneous anaphylaxis (PCA) mouse model in vivo. The cell degranulation of RBL-2H3 cells induced by DNP-IgE/DNP-BSA and Compound 48/80 (C48/80) was investigated, and the molecules of degranulation related signaling pathway was further detected by qRT-PCR and Western Blot analysis. Meanwhile, therapeutic effect of SD Decoction and POG were evaluated using PCA models in vivo. The molecular docking technology was conducted to explore the potential mechanisms. ResultsIn cells model induced by DNP-IgE/DNP-BSA, the release rate of β-Hex in high dose of SD and POG groups were 43.79% and 57.01%, and the release amount of HA in high dose of SD and POG groups were 26.19 ng/mL and 24.20 ng/mL. They were significantly lower than that in the model group. Besides, SD decoction and POG could significantly inhibit intracellular Ca2+ increasing and cell apoptosis. But there is no obvious effect on cells degranulation induced by C48/80. The molecular docking results showed that 5-O-Methylvisamioside and POG could bind with FcεRI α with stronger binding ability, but weak binding ability to Mrgprx2. Moreover, qPCR and Western blot analyses indicated that SD could down-regulate Lyn/Syk/PLCγ, MAPK and PI3K/AKT/NF-κB signal pathway to inhibit IgE-dependent cell degranulation. In mice PCA model, both SD and POG could dose-dependently attenuate the Evans Blue extravasation, paw and ear swelling induced by DNP-IgE/DNP-BSA, but no significant inhibition under the PCA models induced by C48/80. ConclusionIn conclusion, SD is effective for the therapeutic of type I allergies, suggesting that SD is a potential candidate for the treatment of type I allergy, and the underlying mechanism of these effects needs to be further studied.

Isolation and Quantification of 2-O-Caffeoyl-hydroxycitric Acid, an Active Component in Hot Water Extracts of Coriander (Coriandrum sativum L.), Which Inhibits Degranulation of RBL-2H3 Cells

Objectives: Natural compounds that can effectively prevent and alleviate allergic symptoms, such as hay fever, are widely sought after from sources such as natural ingredients that can be ingested daily. Coriander extracts have been reported to show anti-allergic activity; however, hot water extracts have not been tested previously. Here, we found that a hot water extract of the aerial parts of coriander inhibited the degranulation of rat basophilic leukemia cell line RBL-2H3 cells and identified the active compounds. Methods: Chromatographic methods were used to isolate compounds from the hot water extract of coriander. The chemical structures were elucidated by analyses of high-resolution electrospray ionization mass spectrum and nuclear magnetic resonance spectral data. The degranulation activity of compounds was evaluated using the β-hexosaminidase release assay. Results: We analyzed the components to identify active compounds. 2 S,3 S-2- O-caffeoyl-hydroxycitric acid (1), and 2- O-caffeoyl-hydroxycitric acid 6′- O-methyl ester (2) were isolated from the hot water extract of coriander, and both compounds showed degranulation inhibitory activity in RBL-2H3 cells at a concentration below 2.5 mM. The content of compound 1 in the freeze-dried coriander powder was calculated to be 2.0% via HPLC quantification. Conclusions: Compound 1 was considered to be the primary active contributor in the hot water extract of coriander.

Effects of Simvastatin on RBL-2H3 Cell Degranulation.

Hypercholesterolemia is a major complication of arteriosclerosis. Mast cells in arteriosclerosis plaques induce inflammatory reactions and promote arterial sclerosis. In this study, we evaluated the pharmacological effects of simvastatin (SV)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors on the degranulation of rat basophilic leukemia (RBL)-2H3 cells, which are commonly used as mast cell models. SV significantly decreased the degranulation induced by three types of stimulation: antigen antibody reaction (Ag-Ab), thapsigargin (Tg) serosal endoplasmic reticulum calcium ATPase (SERCA) inhibitor, and A23187 calcium ionophore. SV had a stronger inhibitory effect on degranulation induced by Ag-Ab stimulation than the other two stimulations. However, SV did not inhibit increase of intracellular Ca2+ concentrations. Mevalonate or geranylgeraniol co-treatment with SV completely prevented the inhibitory effect of SV on the degranulation induced by these stimulations. Immunoblotting results showed that SV inhibited protein kinase C (PKC) delta translocation induced by Ag-Ab but not by Tg or A23187. SV induced a reduction in active Rac1, and actin filament rearrangement. In conclusion, SV inhibits RBL-2H3 cell degranulation by inhibiting downstream signaling pathways, including the sequential degranulation pathway. These inhibitory effects were completely reversed by the addition of geranylgeraniol and might be induced by changes in the translocation of the small guanosine 5'-triphosphatase (GTPase) families Rab and Rho, which are related to vesicular transport PKC delta translocation and actin filament formation, respectively. These changes are caused by the inhibition of HMG-CoA reductase by SV following the synthesis of geranylgeranyl pyrophosphates, which play important roles in the activation of small GTPases, Rab.

Assessment of the effect of glycation on the allergenicity of sesame proteins

Sesame allergy is a growing concern worldwide. In this study, sesame proteins was glycated with glucose, galactose, lactose and sucrose respectively, and the allergenicity of different glycated sesame proteins were assessed by a comprehensive strategy, including simulated gastrointestinal digestion in vitro, a BALB/c mice model, a rat basophilic leukemia (RBL)–2H3 cell degranulation model and a serological experiment. Firstly, simulated gastrointestinal digestion in vitro showed that glycated sesame proteins were more easily to digest than raw sesame. Subsequently, the allergenicity of sesame proteins was assessed in vivo by detecting the allergic indexes of mice, and results showed that the levels of total immunoglobulin E (IgE) and histamine were reduced in glycated sesame proteins treated mice. Meanwhile, the Th2 cytokines (IL-4, IL-5, and IL-13) were downregulated significantly, demonstrating that sesame allergy was relieved in glycated sesame treated mice. Thirdly, the RBL-2H3 cell degranulation model results showed that the release of β-hexosaminidase and histamine were decreased to different degrees in glycated sesame proteins treated groups. Notably, the monosaccharide glycated sesame proteins exhibited lower allergenicity both in vivo and in vitro. Furthermore, the study also analyzed the structure alteration of sesame proteins, and the results showed that the secondary structure of glycated sesame proteins were changed (the content of α-helix and β-sheet were reduced), and the tertiary structure of sesame proteins after glycation modification was also changed (microenvironment around aromatic amino acids was altered). Besides, the surface hydrophobicity of glycated sesame proteins was also reduced except sucrose glycated sesame proteins. In conclusion, this study demonstrated that glycation reduced the allergenicity of sesame proteins effectively, especially glycation with monosaccharides, and the allergenicity reduction might be related to structural changes. The results will provide a new reference for developing hypoallergenic sesame products.

Synthesis and structure-activity relationship of kujigamberol B, a dinorlabdane diterpenoid isolated from an ancient Kuji amber.

The versatile methodology was developed for synthesizing kujigamberol B, a dinorlabdane diterpenoid isolated from the methanol extract of Kuji amber. A highly efficient intramolecular cyclization is followed by a Sonogashira-coupling reaction during the total synthesis. The synthesized compounds were evaluated for the growth-restoring activity against the mutant yeast (zds1Δ erg3Δ pdr1Δ pdr3Δ) and for the degranulation of RBL-2H3 cells. We found that in both activities, primary alcohol and secondary alcohol analogs are as active as kujigamberol B.

Effects of Lactobacillus fermentation on Eucheuma spinosum polysaccharides: Characterization and mast cell membrane stabilizing activity

Eucheuma polysaccharides have varieties of biological activities. However, it is accompanied by problems like large molecular weight, high viscosity, and low utilization. Here, we first prepared fermented Eucheuma spinosum polysaccharides (F-ESP) by Lactobacillus fermentation, compared with low-temperature freeze-thaw ESP (L-ESP) prepared by the freeze-thaw method, explored the composition and structural characteristics of F-ESP and L-ESP, and evaluation of the ability of different samples to inhibit mast cell degranulation using classical mast cell model. Then, the activity of L-ESP and F-ESP in vivo was preliminarily evaluated using a passive cutaneous anaphylaxis model. Two kinds of F-ESP named F1-ESP-3 and F2-ESP-3 were obtained by fermentation of Eucheuma spinosum with the selected strains of Lactobacillus.sakei subsp.sakei and Lactobacillus.rhamnosus. Compared with the purified component L-ESP-3, the monosaccharide composition of F1-ESP-3 contains more glucuronic acid, the molecular weight reduced from >600 kDa (L-ESP-3) to 28.30 kDa (F1-ESP-3) and 33.58 kDa (F2-ESP-3), F1-ESP-3 has higher solubility and lower apparent viscosity. Fermentation did not destroy the functional groups and structure of ESP. Moreover, F1-ESP-3 significantly inhibited RBL-2H3 cell degranulation by reducing depolymerization of F-actin and Ca2+ influx. F1-ESP-3 reduced the symptoms of mast cell-mediated passive cutaneous anaphylaxis, indicating that F1-ESP-3 may have better anti-allergic activity in vivo.

Ultramicronized N-Palmitoylethanolamine Regulates Mast Cell-Astrocyte Crosstalk: A New Potential Mechanism Underlying the Inhibition of Morphine Tolerance.

Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 μM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 μM, 18 h), as evaluated by β-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1β, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk.

Sea cucumber chondroitin sulfate polysaccharides attenuate OVA-induced food allergy in BALB/c mice associated with gut microbiota metabolism and Treg cell differentiation.

Previous research studies have shown that sulfated polysaccharides can inhibit food allergy, but the detailed mechanism remains largely unknown. In this study, RBL-2H3 cells were used to compare the anti-allergic activities of four sulfated polysaccharides, and an ovalbumin (OVA)-sensitized allergic mouse experiment was used to explore their desensitization effect, with regard to the alteration in gut microbiota and immune cell differentiation. Compared with the shark, bovine and porcine chondroitin sulfate, sea cucumber chondroitin sulfate (SCCS) significantly inhibited the degranulation of RBL-2H3 cells. SCCS reduced allergic symptoms and protected the jejunum from injury in mice. Furthermore, SCCS increased the relative abundance of Lachnospiraceae NK4A136, decreased the relative proportion of Prevotellaceae NK3B31, and up-regulated the secretion of short chain fatty acids such as butyric acid in the feces, resulting in an increase in the mucin 2 (MUC2) secretion by goblet cells HT-29. Meanwhile, SCCS induced the differentiation of regulatory T cells in the mesenteric lymph nodes of mice. This study provides a deeper understanding of the functioning mechanism of SCCS in alleviating food allergy and may guide the development and production of anti-allergy active ingredients.

The Heterogeneity of Ovomucoid-Specific IgE Idiotype Is Associated With Egg Allergy Symptom Severity.

Immunoglobulin E (IgE)-mediated egg allergy presents as one of the most common food allergies. The level of specific IgE (sIgE) antibody is widely used as an important in vitro diagnostic indicator. However, sIgE antibody levels are often inconsistent with the clinical manifestations of patients. The heterogeneity of egg-specific IgE idiotypes (sIgE-IDs) may help reflect clinical egg allergy severity. Eight peptides were synthesized, corresponding to the linear epitopes of ovomucoid (OVM). The sIgE-IDs of egg-allergic patients were detected by enzyme-linked immunosorbent assay. Fresh peripheral blood was collected from patients with different heterogeneity strength of sIgE-ID, and egg extract was used as a stimulus to the basophil activation test (BAT). RBL-2H3 cells were sensitized with serum with different strength of sIgE-ID heterogeneity and the release rate of β-hexosaminidase was calculated. Among 75 patients with egg allergy, 24% had sIgE for all epitopes and 85% had sIgE for at least one epitope. Analysis of individual patients revealed differences in epitope recognition patterns among the patients, that is, heterogeneity in sIgE-ID. More importantly, the number of IgE-positive peptides had a strong correlation with allergic symptoms in egg-allergic patients (r = 0.706). BAT and RBL-2H3 cell degranulation confirmed that higher sIgE-ID heterogeneity strength was more effective in inducing effector cell responses. Our results suggest that the greater the heterogeneity strength of OVM-sIgE-ID, the more severe the allergic symptoms.

Protection of Allergic Asthma in Mice by Black Rice Bran Bioprocessed with Shiitake Mushroom Mycelia

We evaluated the potential of orally fed new food formulations to inhibit biomarkers reported to be involved in the causes of allergic asthma in mice. Asthma, a serious non-communicable disease, affects both adults and children and can be undertreated. New functional foods could provide therapeutic approaches. Here, the anti-asthma mechanism of a new functional food and three isolated fractions produced by bioprocessing black rice bran with shiitake mushroom mycelia was evaluated in mast cells, B cells, and orally fed mice and compared with non-bioprocessed black rice bran. In vitro, the treatments inhibited RBL-2H3 cell degranulation and immunoglobulin E (IgE) production. The in vitro anti-asthma effects were confirmed in orally fed mice following asthma induction by alumina and chicken egg ovalbumin (OVA). The suppression of asthma resulted from the inhibition of inflammation- and immune-related substances, including OVA-specific IgE, thymic stromal lymphopoietin, eotaxin, leukotriene C4, prostaglandin D2, and vascular cell adhesion molecule-1 in bronchoalveolar lavage fluid and serum. The treatment also reversed the thickening of the lung airway wall. The inflammation and asthma inhibition seems to be regulated by the balance of the T-helper cells’ Th1/Th2 immune response and the inhibition of multiple biomarkers associated with the cause of asthma. Future human clinical studies with adults and children should determine the potential therapeutic value of the anti-asthma effects of the new functional foods.

Aqueous Extract from Cuminum cyminum L. Seed Alleviates Ovalbumin-Induced Allergic Rhinitis in Mouse via Balancing of Helper T Cells

Cuminum cyminum L. (cumin) seeds are widely used as a spice. Although we previously reported that the aqueous extract of cumin seeds suppresses the degranulation of rat basophilic RBL-2H3 cells, it has not been clarified whether the extract alleviates actual allergy symptoms in vivo. Therefore, in this study, we investigated the effect of oral administration of cumin seed aqueous extract (CAE) in ovalbumin (OVA)-induced allergic rhinitis. BALB/c mice were randomly divided into the following three groups: control group (five mice), OVA group (five mice), and OVA + CAE group (five mice). Allergic rhinitis was induced by sensitization (intraperitoneal, 25 μg OVA and 1.98 mg aluminum hydroxide gel) followed by challenge (intranasal, 400 μg OVA). The oral administration of CAE (25 mg/kg) reduced the sneezing frequency of OVA-induced allergic rhinitis model mice. In addition to reducing the serum immunoglobulin E and IL-4 levels, the oral administration of CAE reduced the production of T-helper type-2 (Th2) cytokines (IL-4, IL-5, IL-10, and IL-13) in the splenocytes of the model mice. Furthermore, a significant increase in the ratio of Th1 to Th2 cells was observed in the CAE-administered group. Our findings suggest that the ingestion of CAE improves T cell balance, the dominant state of Th2, and alleviates allergic rhinitis symptoms.

The amino acids differences in epitopes may promote the different allergenicity of ovomucoid derived from hen eggs and quail eggs

Quail egg ovomucoid can inhibit activation of basophils and eosinophils, while hen egg ovomucoid has been shown to be a major allergen, named Gal d 1. At present, the differences in structure and function between two ovomucoid are unclear. We found the homology of ovomucoid in quail eggs and hen eggs reached 77 %. Compared with hen egg ovomucoid, the distribution of secondary structure was different in AA52−53, AA57−58, AA66−68, AA71−72, AA131−133, AA139−140, AA157−159 and AA184−185. Among 9 epitopes of egg ovomucoid, there were different amino acids from quail egg ovomucoid in 8 epitopes. Recombination quail egg ovomucoid had trypsin inhibition activity and quail egg ovomucoid didn’t specifically bind to serum of eggs allergic patients. Quail egg ovomucoid can significantly inhibit RBL-2H3 cells degranulation and protect cells morphology to a certain extent, indicating quail egg ovomucoid can inhibit cells activation and have potential anti-allergic effects, which is related to trypsin inhibitory activity. The difference in sensitization compare to hen egg ovomucoid may be due to amino acids differences affecting protein structure by changing antigenic epitopes.

GRK2 inhibitors, paroxetine and CCG258747, attenuate IgE-mediated anaphylaxis but activate mast cells via MRGPRX2 and MRGPRB2.

G protein-coupled receptor (GPCR) kinase 2 (GRK2), which phosphorylates agonist-occupied GPCRs to promote their desensitization, has been investigated as an attractive therapeutic target for cardiovascular and metabolic diseases. Several GRK2-targeted inhibition strategies have been reported including the use of direct pharmacological inhibitors such as paroxetine (a widely prescribed antidepressant) and its analogs such as compound CCG258747. Cross-linking of high affinity IgE receptor (FcϵRI) on mast cells (MCs) and the resulting degranulation causes anaphylaxis and allergic asthma. Using gene silencing strategy, we recently showed that GRK2 contributes to FcεRI signaling and MC degranulation. The purpose of this study was to determine if the GRK2 inhibitors paroxetine and CCG258747 modulate FcεRI-mediated MC responses in vitro and in vivo. Utilizing rat basophilic leukemia (RBL-2H3) cells and primary mouse lung MCs (LMCs), we found that paroxetine and CCG258747 inhibit FcϵRI-mediated calcium mobilization and degranulation. Furthermore, intravenous administration of paroxetine and CCG258747 in mice resulted in substantial reduction of IgE-mediated passive cutaneous anaphylaxis. Unlike LMCs, human cutaneous MCs abundantly express a novel GPCR known as MRGPRX2 (mouse; MRGPRB2). We found that in contrast to their inhibitory effects on FcεRI-mediated MC responses, both paroxetine and CCG258747 induce calcium mobilization and degranulation in RBL-2H3 cells stably expressing MRGPRX2 but not in untransfected cells. Furthermore, paroxetine and CCG258747 induced degranulation in peritoneal MCs from Wild-type (WT) mice in vitro and caused increased cutaneous vascular permeability in vivo, but these responses were substantially reduced in Mrgprb2−/− mice. Additionally, upon intradermal injection, paroxetine also induced neutrophil recruitment in WT but not Mrgprb2−/− mice. These findings suggest that in addition to their potential therapeutic utility against cardiovascular and metabolic disorders, paroxetine-based GRK2-inhibitors may serve to modulate IgE-mediated anaphylaxis and to enhance cutaneous host defense by harnessing MC’s immunomodulatory property through the activation of MRGPRX2/MRGPRB2.

Inhibition of MRGPRX2 but not FcεRI or MrgprB2-mediated mast cell degranulation by a small molecule inverse receptor agonist.

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) contributes to hypersensitivity reactions to cationic US-Food and Drug Administration (FDA) approved drugs such as the neuromuscular blocking agent, rocuronium. In addition, activation of MRGPRX2 by the neuropeptide substance P (SP) and the pro-adrenomedullin peptide (PAMP-12) is associated with a variety of cutaneous conditions such as neurogenic inflammation, pain, atopic dermatitis, urticaria, and itch. Thus, small molecules aimed at blocking MRGPRX2 constitute potential options for modulating IgE-independent MC-mediated disorders. Two inverse MRGPRX2 agonists, named C9 and C9-6, have recently been identified, which inhibit basal G protein activation and agonist-induced calcium mobilization in transfected HEK293 cells. Substance P serves as a balanced agonist for MRGPRX2 whereby it activates both G protein-mediated degranulation and β-arrestin-mediated receptor internalization. The purpose of this study was to determine if C9 blocks MRGPRX2’s G protein and β-arrestin-mediated signaling and to determine its specificity. We found that C9, but not its inactive analog C7, inhibited degranulation in RBL-2H3 cells stably expressing MRGPRX2 in response to SP, PAMP-12 and rocuronium with an IC50 value of ~300 nM. C9 also inhibited degranulation as measured by cell surface expression of CD63, CD107a and β-hexosaminidase release in LAD2 cells and human skin-derived MCs in response to SP but not the anaphylatoxin, C3a or FcϵRI-aggregation. Furthermore, C9 inhibited β-arrestin recruitment and MRGPRX2 internalization in response to SP and PAMP-12. We found that a G protein-coupling defective missense MRGPRX2 variant (V282M) displays constitutive activity for β-arrestin recruitment, and that this response was significantly inhibited by C9. Rocuronium, SP and PAMP-12 caused degranulation in mouse peritoneal MCs and these responses were abolished in the absence of MrgprB2 or cells treated with pertussis toxin but C9 had no effect. These findings suggest that C9 could provide an important framework for developing novel therapeutic approaches for the treatment of IgE-independent MC-mediated drug hypersensitivity and cutaneous disorders.

Identification of components in coriander (Coriandrum sativum L.) inhibiting degranulation of RBL-2H3 cells

We found that a water-soluble extract of coriander (Coriandrum sativum L.) (leaves, petioles and stems) inhibits antigen-induced degranulation of RBL-2H3 cells, a rat basophil leukemia cell line. The aim of this study was to elucidate the anti-degranulation active components in the extract. The methanol-eluate fraction obtained by fractionation of the water-soluble extract using MCI gel column chromatography had strong activity, and eight components were isolated and identified. Two of them were identified as new compounds, (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucopyranoside (compound 1) and (7S,8R)-7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-fro[2,3-g][2]benzopyran-5-one (compound 2). As a result of evaluation of anti-degranulation activity of eight components, seven of them, such as tryptophan, phenylalanine, dihydroxycoumarin glucoside, quercetin glycoside, rutin, compound 1, and compound 2, had the activity. These results indicated that the water-soluble extract of coriander contains several anti-degranulation substances.

Exploring the mechanism of sweetener neohesperidin dihydrochalcone on oral tolerance via a network pharmacology approach combined with vivo and vitro methods

Neohesperidin dihydrochalcone (NHDC) is a kind of sweetener widely used in food processing. Some artificial sweeteners induce food allergy by breaking oral tolerance (OT). The relationship between NHDC and oral tolerance are important in food safety. We explored the possible effects of NHDC on OT via network pharmacology combined with vivo and vitro experiments. NHDC was predicted to have effects on OT. It was focus on immunoglobulin production, inflammation and immunity. Then, OT mouse model and mast cell degranulation were applied to further explore the mechanism. NHDC could affect the Th2 response by suppressing the sIgG1 level in the serum and GATA-3 expression in the colon. NHDC could inhibit RBL-2H3 cells degranulation by suppressing the Ca2+ influx and MAPK signaling pathways. Results showed that NHDC can be safely applied in food processing and had promotional effects on oral tolerance.

Anti-Allergic Effect of Aqueous Extract of Coriander (Coriandrum sativum L.) Leaf in RBL-2H3 Cells and Cedar Pollinosis Model Mice

Coriander (Coriandrum sativum L.) is classified in the Apiaceae family and used as an herb. Coriander leaf has been reported to possess various health functions. Here, we report the anti-allergic effect of aqueous coriander leaf extract (ACLE). ACLE with 1.0 mg/mL or higher concentration significantly inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner with no cytotoxicity. ACLE suppressed the increase in the intracellular Ca2+ concentration in response to antigen-specific stimulation. Immunoblot analysis demonstrated that ACLE significantly downregulates phosphorylation of phosphatidylinositol 3-kinase and tends to downregulate phosphorylation of Syk kinase in the signaling pathways activated by antigen-mediated stimulation. Oral administration of ACLE did not alter the sneezing frequency of pollinosis model mice stimulated with cedar pollen, but significantly reduced the serum IgE level. Our data show anti-allergic effects of coriander leaf in both cultured cells and pollinosis mice. These results suggest that coriander leaf has the potential to be a functional foodstuff with anti-allergy effects.