Calceolarioside B alleviates airway barrier dysfunction and inflammation via targeting P2Y₆R in OVA-triggered asthma

Asthma is a chronic inflammatory airway disease characterized by barrier dysfunction and excessive inflammatory response. In this study, we identified that a traditional Chinese medicine Akebia quinate extract alleviated asthmatic phenotype in vivo. Transcriptomic analysis revealed that Akebia quinate extract associated with inflammatory response and airway epithelial barrier remodeling. Using network pharmacology screened the top four active ingredients from Akebia quinate and further evaluated their effects on inflammatory cytokines and airway barrier indicators. Among them, compound Calceolarioside B exhibited the most potent anti-inflammatory and barrier-protective activities. Pharmacodynamic validation confirmed that Calceolarioside B alone significantly ameliorated asthmatic features. The candidate target of Calceolarioside B was identified as P2Y₆R using AI-assisted deep learning, virtual target fishing, and molecular dynamics simulations. In vitro binding assays validated the interaction between Calceolarioside B and P2Y₆R. Furthermore, Calceolarioside B treatment failed to produce additional therapeutic effects in the P2Y₆R knockout asthmatic mouse model, confirming that P2Y₆R was the functionally relevant target of Calceolarioside B. Subsequent molecular docking and point mutation experiments identified LYS-25 as the effective binding site for Calceolarioside B and P2Y₆R. Collectively, these findings demonstrate that Calceolarioside B alleviates asthma by targeting P2Y₆R, providing a mechanistic basis for its therapeutic potential and a rationale for developing target-specific interventions.