ETHNOPHARMACOLOGICAL RELEVANCEZuojin Pill (ZJP), a traditional Chinese medicinal formula, is widely recognized for its diverse pharmacological properties in the management of gastrointestinal disorders. However, the precise mechanisms underlying its therapeutic effects in gastroesophageal reflux disease (GERD) remain inadequately understood.AIM OF THE STUDYThis study aims to investigate the therapeutic effects of ZJP in GERD and to elucidate the molecular mechanisms involved.MATERIALS AND METHODSThe chemical composition of ZJP was characterized using HPLC-Q-Exactive-MS. A rat model of GERD was established through esophagogastric anastomosis, and three different doses of ZJP were administered. Histological changes were assessed via hematoxylin-eosin (H&E) staining, while pro-inflammatory cytokines were quantified to evaluate the anti-inflammatory effects of ZJP. Network pharmacology combined with bioinformatics analysis was employed to predict potential therapeutic targets and signaling pathways of ZJP in GERD. Validation of the mechanisms was conducted through Western blotting, immunofluorescence (IF), transmission electron microscopy (TEM), and immunohistochemistry (IHC).RESULTSThe results demonstrated that ZJP effectively alleviated pathological alterations and reduced pro-inflammatory cytokine levels in esophageal tissues of GERD rats. Western blotting and IF analysis of E-cadherin and claudin-1 confirmed that ZJP enhanced the integrity of the esophageal mucosal barrier. TEM imaging revealed that ZJP restored intercellular space (DIS), increased desmosome density, thereby protecting esophageal tissues from the detrimental effects of GERD. Furthermore, ZJP modulated macrophage polarization in the GERD rat model. Mechanistic investigations indicated that ZJP exerted its therapeutic effects by inhibiting MAPK/NF-κB signaling pathway activation and downregulating the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinase 2 (MMP2), consistent with predictions from network pharmacology analysis.CONCLUSIONSThis study provides comprehensive evidence for the therapeutic efficacy of ZJP in GERD, acting through modulation of inflammation, mucosal barrier integrity, and macrophage polarization. Additionally, ZJP downregulated PTGS2 and MMP2 expression and suppressed the activation of MAPK/NF-κB signaling pathways, underscoring its potential as a therapeutic intervention for GERD.