Discovery of scutellarin as a MRGX2 agonist via MRGX2/CMC strategy and revelation of its pseudo-allergic mechanism

Background Shu-Gan-Ning injection (SGN) is widely used clinically to treat hepatopathy, improve liver function, and enhance immunity. However, pseudo-allergic reactions (PARs) occur after administration and the causative compounds remain unclear. Therefore, it is of paramount importance to pinpoint the “culprit” compound in SGN and explore its underling mechanism. Methods Mas-related G protein-coupled receptor X2 cell membrane chromatography (MRGX2/CMC)-HPLC-MS was established and used to “capture” and identify risk components in SGN. The interactions between risk components and MRGX2 were investigated by integrating MRGX2/CMC detection, virtual docking, and frontal analysis assays. MRGX2-knockdown LAD2 cell models were used to verify the specific target of the risk components and the signaling pathways that trigger PARs. C57BL/6 mice were used to evaluate the PARs induced by hazardous compounds. The conditional knockout mice model was used to evaluate the key role of MAS-related G protein-coupled receptor, member B2 (MRGB2) in mast cells in vivo pseudo-allergic reaction induced by target compound. Results We identified scutellarin, baicalin, and oroxyloside as MRGX2‐binding components in SGN. Affinity analysis revealed K D values of 2.69 × 10 −7 M for scutellarin, 2.18 × 10 −7 M for baicalin, and 7.21 × 10 −7 M for oroxyloside. Functional assays showed that only scutellarin (5–100 μM) concentration-dependently induced mast cell degranulation, Ca 2+ influx, and release of inflammatory mediators. MRGX2 knockdown (mRNA ∼50%, protein ∼70%) significantly attenuated these responses, and pathway analysis confirmed that scutellarin activates the PLCγ/PKC cascade via MRGX2. In vivo , scutellarin induced local paw swelling and Evans blue extravasation, as well as systemic hypothermia, with EC 50 values of 0.92 mg/kg for temperature reduction and 1.82 mg/kg for Evans blue exudation. In mast cell‐specific Mrgb2 conditional knockout (CKO) mice, scutellarin‐induced paw swelling and Evans blue leakage were significantly reduced compared with wild‐type mice, confirming the critical role of MRGB2 in mediating the pseudo‐allergic response. Discussion Collectively, these findings identify scutellarin as an MRGX2 agonist and a pseudo‐allergic risk compound in SGN, highlighting the need for quality control measures for scutellarin in baicalin‐containing preparations.