Mechanism and components of Hewei Jiangni Prescription in regulating MrgprX2/B2 to attenuate MC/DRG neuroimmune dysregulation and mitigate non-erosive reflux disease-associated esophageal hypersensitivity

The Hewei Jiangni Prescription (HP) is a traditional Chinese prescription, which has shown clinical benefits for many years in non-erosive reflux disease (NERD)-associated esophageal hypersensitivity. However, its mechanism of effects remains unclear. To investigate the mechanism of HP in treating NERD-associated esophageal hypersensitivity and identify its pharmacologically active components. NERD-associated esophageal hypersensitivity model mice were treated with HP. Proteomics and protein interaction networks were employed to identify differentially regulated targets. Effects on key targets were validated in cell models and model mice using enzyme-linked immunosorbent assay, western blotting, reverse transcription‒quantitative polymerase chain reaction, and immunofluorescence. Bio-layer interference technology was used to identify HP components binding to targets, followed by liquid chromatography‒mass spectrometry identification and functional validation in cell models. HP mitigated esophageal pathology and visceral hypersensitivity in NERD-associated esophageal hypersensitivity model mice. Additionally, HP may attenuate neuroimmune dysregulation in the experimental model by downregulating MrgprX2/B2 overexpression in mast cells and dorsal root ganglion (DRG) neurons, lowering intracellular Ca 2+ levels, and decreasing the release of mast cell tryptase and calcitonin gene-related peptide. Vitexin was identified as one of the bioactive components of HP, reproducing its core pharmacological effects in vitro and exhibiting high affinity for the MRGX2 target. This study suggests that HP may attenuate neuroimmune dysregulation between mast cells and DRG neurons by regulating MrgprX2/B2, thereby mitigating NERD-associated esophageal hypersensitivity. Additionally, the study findings suggest that vitexin may be one of the bioactive components of HP. HP, Hewei Jiangni Prescription. Green arrow, After the intervention of HP, HP drug serum or vitexin, the corresponding indicator was adjusted downward. MC, mast cell. MrgprX2/B2, MAS-related G protein-coupled receptor X2/B2. MCT, mast cell tryptase. PAR2, protease activated receptor. CGRP, calcitonin gene-related peptide. BLI, bio-layer Interferometry. DRGn, dorsal root ganglion neuron. LC-MS, liquid chromatography-mass spectrometry. • HP mitigates NERD-associated esophageal hypersensitivity • HP attenuates MC/DRG neuroimmune dysregulation via MrgprX2/B2 • HP reduces Ca 2+ mobilization and MCT/CGRP release • Proteomics and PPI identify MrgprX2/B2 as a potential target • Vitexin is one of HP bioactive components targeting MrgprX2/B2