Tao Zhang

Mas-related G-protein-coupled receptor X2 (MRGPRX2) plays a critical role in mast cell activation and the mediation of pseudo-allergic reactions, and its detection in peripheral blood is important for investigating mast cell–related disorders such as allergic rhinitis (AR), chronic spontaneous urticaria (CSU), and asthma. However, existing assays based on polyclonal and hybridoma-derived monoclonal antibodies suffer from limitations in accuracy, reproducibility, and standardization, highlighting the need for alternative antibody strategies. In this study, animals were immunized with a rationally selected MRGPRX2 peptide identified using the IEDB Analysis Resource, and sequence-defined recombinant monoclonal antibodies were generated. A sandwich ELISA was subsequently established and validated using serum samples from patients with AR, CSU, and healthy controls (Chinese Clinical Trial Registry: ChiCTR2400082024, ChiCTR2400094130). The peptide spanning residues 289–330 was identified as an optimal immunogen, and six recombinant antibodies were obtained, among which optimal capture–detection pairs exhibited high affinity as confirmed by surface plasmon resonance and Western blot analysis. The developed ELISA demonstrated excellent linearity over the concentration range of 1.5625–200 ng/mL (y = 82.772x − 9.6566, R² = 0.9996), with satisfactory recovery (83.82–115.60%), low imprecision (within-run CV = 3.9%, between-run CV = 5.8%), and a detection limit of 0.981 ng/mL. In addition, the assay showed good stability and specificity. Serum MRGPRX2 levels were significantly elevated in patients with AR and CSU compared with healthy controls. Collectively, these results demonstrate that the recombinant antibody–based ELISA provides a sensitive, specific, stable, and reproducible platform for circulating MRGPRX2 detection, with promising potential for clinical and translational applications in mast cell–related disorders. Graphical abstract

Serum‐specific IgE (sIgE) and skin prick testing (SPT) have limitations in diagnosing allergic rhinitis (AR). CD63, a mast cell activation biomarker reflecting degranulation extent, has unclear application in AR diagnosis. This study developed a functional mast cell activation test (MAT) for the in vitro diagnosis of AR by quantifying CD63 expression on LAD2 cells stimulated with patient serum and specific allergens. The assay was evaluated in 128 AR patients and 115 controls. The assay showed good reproducibility (CV = 8.26%). In the test cohort, AR patients had significantly higher CD63‐positive rates than controls (5.21% ± 4.55% vs. 1.39% ± 1.79%, p < 0.001). ROC analysis yielded an AUC of 0.84, with 94.60% specificity and 77.10% accuracy at a 2.09% cutoff, which was confirmed in the validation cohort (specificity 90.24%, accuracy 80.51%). In the overall population, CD63 AUC (0.85) was higher than that for histamine (0.68) and total IgE (tIgE, 0.83). The CD63‐positive rate correlated strongly with sIgE ( r = 0.59, p < 0.001) and total IgE ( r = 0.42, p < 0.001), whereas its correlation with histamine release was weaker ( r = 0.35 vs. sIgE). These findings support MAT based on mast cell CD63 as a precise functional tool for AR diagnosis.

Purpose: Mas-related G-protein-coupled receptor member X2 (MRGPRX2) is a key receptor in mast cell activation and plays a critical role in mediating pseudo-allergic reactions. However, its role in allergic rhinitis (AR) remains poorly understood. Therefore, we investigated the correlation between plasma MRGPRX2 and symptoms in AR patients. Patients and Methods: A total of 116 patients with typical AR symptoms and positive skin prick test results and 100 healthy controls were recruited. Plasma MRGPRX2, total and specific IgE, and histamine were measured. AR patients who tested negative for both total IgE and sIgE were defined as IgE-negative. Symptom severity was evaluated using a questionnaire combining the Total Nasal Symptom Score (TNSS) and the Visual Analog Scale (VAS). TNSS assessed nasal (sneezing, rhinorrhea, congestion, itching), each scored from 0 (none) to 3 (severe). The VAS ranged from 0 cm (no symptoms) to 10 cm (most severe). Results: Plasma MRGPRX2 levels were significantly elevated in patients with AR compared with healthy controls (P< 0.001) and demonstrated good discriminative performance (AUC=0.92, P< 0.001). Moreover, patients with moderate to severe AR had significantly higher plasma MRGPRX2 levels than those with mild AR (P< 0.001). A significant positive correlation was also observed between plasma MRGPRX2 levels and VAS scores (ρ=0.37, P< 0.001). This correlation was stronger in the IgE-negative AR group (ρ=0.61, P< 0.001) compared with the IgE-positive group (ρ=0.25, P=0.027) (Z=2.25, P=0.024). Notably, plasma MRGPRX2 levels were positively correlated with TNSS only in IgE-negative AR patients (ρ=0.38, P=0.016), whereas no significant correlation was observed in the IgE-positive group (ρ=0.17, P=0.238). Conclusion: Plasma MRGPRX2 levels are elevated in patients with AR and are positively correlated with disease symptoms, particularly in IgE-negative cases. MRGPRX2 may serve as a novel biomarker to improve the classification and management of AR, especially in patients exhibiting allergic symptoms despite clinically insignificant IgE levels.

Sinomenine hydrochloride (SH) is commonly used in the treatment of rheumatoid arthritis. It activates mast cells and induces anaphylaxis in the clinical setting. Adverse drug reactions can be caused by activation of MAS-associated G protein-coupled receptor X2 (MRGPRX2) on mast cells. Because the ligand binding site of MRGPRX2 is easily contacted in dilute solvents, it can be activated by many opioid drug structures. N-Demethylsinomenine (M-3) has a similar chemical structure to that of the opioid scaffold and is a major metabolite of SH. We sought to clarify whether M-3 induces anaphylaxis synergistically with its prototype in a mouse model. Molecular docking computer simulations suggested a similar binding effect between M-3 and SH. M-3 was chemically synthesized and analyzed by surface plasmon resonance to reveal its affinity for MRGPRX2. Temperature monitoring, in vivo hindlimb swelling and exudation test, and in vitro mast cell degranulation test were used to explore the mechanism of MRGPrx2 mediated allergic reaction triggered by M-3. Reduced M-3-induced inflammation was evident in MrgprB2 (the ortholog of MRGPRX2) conditional (Cpa3-Cre/MrgprB2flox) knockout (MrgprB2-CKO) mice. Additionally, LAD2 human mast cells with MRGPRX2 knockdown showed reduced degranulation. M-3 activated LAD2 cells synergistically with SH as regulated by GRK2 signaling and IP3R/PLC/PKC/P38 molecular signaling pathways. The results indicate that the M-3 metabolite can activate mast cells synergistically with its prototype SH via MRGPRX2 and aggravate anaphylaxis. These findings provide important insights into drug safety.

Mast cells (MCs) are important therapeutic targets for allergic diseases. High‐affinity immunoglobulin E (IgE) Fc receptors (FcεRI) trigger abnormal activation of MCs. Allergic rhinitis (AR) is an IgE‐mediated antigen inhalation reaction that occurs in the nasal mucosa. MC aggravation and dysfunction were observed during the early stages of AR pathogenesis. Herb‐derived dictamnine exhibits anti‐inflammatory effects. Here, we investigated the pharmacological effects of herb‐derived dictamnine on IgE‐induced activation of MCs and an ovalbumin (OVA)‐induced murine AR model. The results indicated that dictamnine attenuated OVA‐induced local allergic reactions and reduced body temperature in OVA‐challenged mice with active systemic anaphylaxis. Additionally, dictamnine decreased the frequency of nasal rubbing and sneezing in an OVA‐induced murine AR model. Moreover, dictamnine inhibited FcεRI‐activated MC activation in a dose‐dependent manner without causing cytotoxicity, reduced the activation of the tyrosine kinase LYN in LAD2 cells, and downregulated the phosphorylation of PLCγ1, IP3R, PKC, Erk1/2, and Akt, which are downstream of LYN. In conclusion, dictamnine suppressed the OVA‐stimulated murine model of AR and activated IgE‐induced MCs via the LYN kinase‐mediated molecular signaling pathway, suggesting that dictamnine may be a promising treatment for AR.

Anaphylaxis is a type of potentially fatal hypersensitivity reaction resulting from the activation of mast cells. Many endogenous or exogenous factors could cause this reaction. Silibinin is the main chemical component of silymarin and has been reported to have pharmacological activities. However, the anti-allergic reaction effect of silibinin has not yet been investigated. This study aimed to evaluate the effect of silibinin to attenuate pseudo-allergic reactions in vivo and to investigate the underlying mechanism in vitro. In this study, calcium imaging was used to assess Ca2+ mobilization. The levels of cytokines and chemokines, released by stimulated mast cells, were measured using enzyme immunoassay kits. The activity of silibinin was evaluated in a mouse model of passive cutaneous anaphylaxis (PCA). Western blotting was used to explore the related molecular signaling pathways. In results, silibinin markedly inhibited mast cell degranulation, calcium mobilization, and preventing the release of cytokines and chemokines in a dose-dependent manner via the PLC gamma and PI3K/Akt signaling pathway. Silibinin also attenuated PCA in a dose-dependent manner. In summary, silibinin has an anti-pseudo-allergic pharmacological activity, which makes it a potential candidate for the development of a novel agent to arrest pseudo-allergic reactions.