Material Basis and MRGPRB3-Associated Mechanisms of Dictamni Cortex Against Pseudo-Allergic Reactions.

ETHNOPHARMACOLOGICAL RELEVANCE: Dictamni Cortex (BXP), derived from the dried root bark of Dictamnus dasycarpus Turcz., is a traditional herbal medicine widely recognized for its anti-inflammatory and anti-allergic properties, particularly in treating skin diseases.Although BXP inhibits mast cell degranulation, its regulatory effects on the pseudo-allergy receptor MRGPRB3 and its primary blood-absorbed active components remain unclear.

AIM OF THE STUDY: This study investigated the in vivo protective effects of BXP against pseudo-allergic reactions, identified its absorbed constituents, and evaluated their effects on MRGPRB3-associated mast-cell activation in vitro.

MATERIALS AND METHODS: Compound 48/80 (C48/80) was used to establish local pseudo-allergic, systemic pseudo-allergic, pruritus, and RBL-2H3 cell degranulation models. UPLC-Q-TOF/MS was used to identify absorbed constituents in BXP-containing serum. Molecular docking, molecular dynamics simulation, and cellular thermal shift assay (CETSA) were used to screen and evaluate potential MRGPRB3-associated active constituents, followed by functional validation using siRNA-mediated Mrgprb3 knockdown. Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells expressing human MRGPRX2 were used to explore the effects of these constituents on MRGPRX2-associated G-protein signaling.

RESULTS: In vivo, BXP reduced C48/80-induced dorsal skin vascular leakage, paw swelling, Evans blue extravasation, systemic hypothermia, and scratching behavior. BXP also alleviated skin histopathological damage and decreased serum monocyte chemoattractant protein-1 (MCP-1), histamine, and tumor necrosis factor-α (TNF-α) levels. In vitro, BXP preserved RBL-2H3 cell morphology, reduced intracellular Ca2+ mobilization and MRGPRB3 expression, and inhibited the release of histamine, β-hexosaminidase (β-Hex), MCP-1, TNF-α, and interleukin-4 (IL-4). UPLC-Q-TOF/MS identified five absorbed constituents in BXP-containing serum: obacunone, fraxinellone (Fra), dictamnine, palmitic acid, and 2(1H)-quinolinone. Molecular docking and molecular dynamics simulations identified Fra as a candidate constituent with potential MRGPRB3-associated activity. BRET assays showed that Fra induced a concentration-dependent change in ΔBRET in HEK293 cells expressing human MRGPRX2, suggesting potential modulation of MRGPRX2-associated G-protein signaling. CETSA showed that Fra increased the thermal stability of a putative MRGPRB3-reactive band in RBL-2H3 lysates, providing preliminary evidence consistent with possible target engagement. Compared with dictamnine, Fra more effectively preserved cell morphology and inhibited MRGPRB3 expression, Ca2+ mobilization, and inflammatory mediator production in vitro. SiRNA-mediated Mrgprb3 knockdown reduced C48/80-induced β-Hex release and MCP-1, TNF-α, and IL-4 mRNA expression. Following Mrgprb3 knockdown, the additional inhibitory effects of Fra on these inflammatory mediators, IP3 generation, and Ca2+ mobilization were weakened. In a separate safety assessment, high-dose BXP altered liver- and kidney-related indicators, whereas Fra did not significantly affect the examined indicators.

CONCLUSIONS: BXP exhibited anti-pseudo-allergic activity in vivo, whereas Fra attenuated mast-cell degranulation and inflammatory responses in vitro through an MRGPRB3-associated IP3/Ca2+ pathway. These findings identify Fra as an absorbed constituent with in vitro activity, while its in vivo anti-pseudo-allergic efficacy requires further investigation.