Integrative phytochemical profiling and systems pharmacology identify ERBB2–MAPK1–AKT1 signaling as a molecular basis for the anti-vertigo effects of Ziyin Zhixuan Decoction

Deciphering the polypharmacological mechanisms of chemically complex therapeutics remains a central challenge in systems biochemistry. Here, we developed an integrated computational–experimental framework to elucidate the molecular basis underlying the anti-vertigo effects of Ziyin Zhixuan Decoction (ZYZX), a multi-component formulation with documented clinical efficacy. Comprehensive chemical profiling by UPLC-Q-TOF-MS/MS identified 64 putative bioactive constituents, predominantly flavonoids, iridoids, and phenolic glycosides. These compounds were systematically mapped onto disease-relevant molecular networks through multi-database target prediction, high-confidence protein–protein interaction (PPI) analysis, and iterative topological screening, which consistently prioritized ERBB2, MAPK1, and AKT1 as central regulatory nodes. To link network-level predictions with physiological outcomes, untargeted serum metabolomics was performed in a thyroxine/reserpine-induced rat model, revealing that ZYZX treatment significantly corrected disruptions in cyclic nucleotide signaling and restored hepato-renal metabolic homeostasis. Molecular docking further supported direct interactions between representative ZYZX constituents and core signaling proteins. Importantly, these computationally inferred mechanisms were experimentally validated by quantitative RT-PCR and Western blotting, demonstrating that ZYZX modulates PI3K–AKT and MAPK signaling pathways at both transcriptional and translational levels, in parallel with improvements in vertigo-like behavioral phenotypes. Collectively, this study provides mechanistic evidence that ZYZX exerts therapeutic effects through coordinated regulation of conserved kinase-driven signaling networks and systemic metabolic pathways. More broadly, it establishes a reproducible, multi-omics-driven strategy for deconvoluting the systems-level actions of polypharmacological interventions, with applicability beyond a single therapeutic context. • Established an integrative systems pharmacology and multi-omics framework for TCM formulas. • Identified key bioactive constituents of Ziyin Zhixuan Decoction via UPLC–Q-TOF-MS/MS. • ERBB2, MAPK1, and AKT1 emerged as convergent hubs across computational and omics layers. • ZYZX alleviates vertigo by co-regulating PI3K–AKT survival and MAPK stress signaling. • In vivo and metabolomic data validated network pharmacology–predicted targets.