Guizhi Wuling Decoction alleviates myocardial fibrosis by restoring mitochondrial homeostasis: Evidence from network pharmacology, molecular docking, and multi-omics integration

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Wuling Decoction (GWD), a combined prescription consisting of the classical formulas Wuling San and Guizhi Fuling Wan without modification, has been clinically applied for cardiovascular diseases. Previous studies have reported its cardioprotective and anti-fibrotic effects; however, the underlying molecular mechanisms remain incompletely understood. AIM OF THE STUDY: To investigate whether restoration of mitochondrial homeostasis contributes to the anti-fibrotic effects of GWD and to explore the involvement of AMPK/PGC-1α signaling. MATERIALS AND METHODS: Myocardial fibrosis (MF) was induced in C57BL/6 J mice by isoproterenol (ISO) administration. Cardiac function, myocardial fibrosis, mitochondrial ultrastructure, mitochondrial membrane potential, ATP production, and NAD+/NADH ratio were evaluated. Absorbed constituents in GWD-containing serum were characterized using UHPLC-MS/MS. Integrated transcriptomic-proteomic analysis highlighted AMPK signaling as one of the important pathways associated with GWD treatment. Molecular docking and molecular dynamics simulations were conducted to evaluate the interactions between representative absorbed compounds and candidate targets. The involvement of AMPK signaling was further validated by pharmacological inhibition using Compound C. RESULTS: GWD significantly improved cardiac function, attenuated myocardial fibrosis, and restored mitochondrial homeostasis, as evidenced by improved mitochondrial ultrastructure, increased mitochondrial membrane potential, ATP production, and NAD+/NADH ratio. Integrated transcriptomic and proteomic analyses identified AMPK signaling as an important pathway associated with the protective effects of GWD. Mechanistically, GWD activated the AMPK/PGC-1α signaling pathway and upregulated mitochondrial biogenesis- and quality control-related proteins, including NRF1, TFAM, and MFN2. Pharmacological inhibition of AMPK by Compound C significantly attenuated GWD-associated improvement of mitochondrial homeostasis and anti-fibrotic effects. CONCLUSION: GWD alleviates ISO-induced MF, at least in part, by restoring mitochondrial homeostasis. Integrated multi-omics analyses and pharmacological validation suggest that AMPK/PGC-1α signaling may contribute to the anti-fibrotic effects of GWD. These findings provide experimental evidence suggesting the involvement of mitochondrial homeostasis in MF and offer new insights into the anti-fibrotic effects of GWD.