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Background: Pseudo-allergic reactions are potentially fatal hypersensitivity responses caused by mast cell activation. α-linolenic acid (ALA) is known for its anti-allergic properties. However, its potential anti-pseudo-allergic effects were not much investigated., Purpose: To investigate the inhibitory effects of ALA on IgE-independent allergy in vitro, and in vivo, as well as the mechanism underlying its effects., Methods/study Designs: The anti-anaphylactoid activity of ALA was evaluated in passive cutaneous anaphylaxis reaction (PCA) and systemic anaphylaxis models. Calcium imaging was used to assess intracellular Ca 2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of Lyn-PLCγ-IP3R-Ca 2+ and Lyn-p38/NF-κB signaling pathway., Results: ALA (0, 1.0, 2.0, and 4.0 mg/kg) dose-dependently reduced serum histamine, chemokine release, vasodilation, eosinophil infiltration, and the percentage of degranulated mast cells in C57BL/6 mice. In addition, ALA (0, 50, 100, and 200 μM) reduced Compound 48/80 (C48/80) (30 μg/ml)-or Substance P (SP) (4 μg/ml)-induced calcium influx, mast cell degranulation and cytokines and chemokine release in Laboratory of Allergic Disease 2 (LAD2) cells via Lyn-PLCγ-IP3R-Ca 2+ and Lyn-p38/NF-κB signaling pathway. Moreover, ALA (0, 50, 100, and 200 μM) inhibited C48/80 (30 μg/ml)- and SP (4 μg/ml)-induced calcium influx in Mas-related G-protein coupled receptor member X2 (MrgX2)-HEK293 cells and in vitro kinase assays confirmed that ALA inhibited the activity of Lyn kinase. In response to 200 μM of ALA, the activity of Lyn kinase by (7.296 ± 0.03751) × 10 -5 units/μl and decreased compared with C48/80 (30 μg/ml) by (8.572 ± 0.1365) ×10 -5 units/μl., Conclusion: Our results demonstrate that ALA might be a potential Lyn kinase inhibitor, which could be used to treat pseudo-allergic reaction-related diseases such as urticaria., (Copyright © 2020. Published by Elsevier GmbH.)

Background: Immunoglobulin E (IgE)-mediated mast cell (MC) activation is crucial in multiple allergic diseases. Parkinson disease protein 7 (DJ-1) and Lyn kinase were reported as the receptor-proximal events in IgE receptor (FcεRI) signals in human MC. Kaempferol, a natural flavonol mainly derived from the rhizome of traditional Chinese herb Kaempferia galanga L. (Zingiberaceae), has been known to inhibit allergic reactions, but it was limited to the receptor-distal signals on rat basophilic leukemia cells. A thorough investigation of the inhibitory effects of kaempferol on human MC has not been done., Purpose: To investigate the inhibitory effects of kaempferol on IgE-mediated anaphylaxis in vivo and in human MCs, as well as the mechanism underlying its effects, especially the receptor-proximal signals., Methods: IgE-mediated passive cutaneous anaphylaxis and systemic anaphylaxis model were applied to elucidate the antiallergic activity of kaempferol in vivo. The degranulation assay, calcium imaging, the release of cytokines and chemokines on the laboratory of allergic disease 2 (LAD2) cells were used to evaluate the antiallergic effect of kaempferol in vitro. Western blot analysis was performed to investigate the DJ-1/Lyn signaling pathway and downstream molecules. Kinase activity assay, immunofluorescence, and molecular docking were conducted to confirm the influence of kaempferol on DJ-1/Lyn molecules., Results: Kaempferol dose-dependently attenuated ovalbumin/IgE-induced mice paw swelling, primary MC activation from paw skin, as well as rehabilitated the hypothermia, and reduced the serum concentrations of histamine, tumor necrosis factor-alpha, interleukin-8, and monocyte chemo-attractant protein-1. Additionally, kaempferol suppressed IgE-mediated LAD2 cell degranulation and calcium fluctuation. Remarkably, kaempferol was found to bind with DJ-1 protein, and initially prevented DJ-1 from translocating to the plasma membrane, thereby inhibited full activation of Lyn, and eventually restrained those receptor-distal signaling molecules, involved Syk, Btk, PLCγ, IP3R, PKC, MAPKs, Akt and NF-κB., Conclusion: Kaempferol could be used as a DJ-1 modulator for preventing MC-mediated allergic disorders through attenuating Lyn activation., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China., Purpose: To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects., Methods/study Designs: The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca 2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2., Results: Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein., Conclusion: Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca 2+ mobilization., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier GmbH. All rights reserved.)