Gouty arthritis (GA), triggered by monosodium urate (MSU) crystal deposition, is closely associated with oxidative stress, mitochondrial dysfunction, and NLRP3 inflammasome-mediated pyroptosis. This study combined network pharmacology, molecular docking, molecular dynamics simulations, and experimental validation to elucidate the mechanism by which the iridoid glycoside ajugol alleviates GA. Network pharmacology analyses identified the PI3K/AKT/mTOR pathway as a key regulatory axis, and docking results revealed stable binding of ajugol to PI3K, AKT, and mTOR. In LPS/MSU-stimulated human chondrocytes, ajugol significantly restored cell viability, reduced LDH release, and preserved extracellular matrix integrity by increasing Collagen II and Aggrecan expression while decreasing MMP-3 levels. Ajugol markedly inhibited the expression of NLRP3, ASC, caspase-1, and GSDMD-N, reduced the secretion of IL-1β, IL-18, IL-6, and TNF-α, and decreased caspase-1 activity and membrane pore formation, indicating that ajugol suppresses pyroptosis through enhanced mitophagy. The mitophagy inhibitor cyclosporin A (CsA) significantly weakened ajugol-induced mitophagy activation and pyroptosis inhibition, confirming that mitophagy plays a pivotal role in its protective effects. Mechanistically, ajugol inhibited PI3K/AKT/mTOR phosphorylation, upregulated PINK1, Parkin, and LC3-II/LC3-I while reducing p62, thereby activating PINK1/Parkin-dependent mitophagy. PI3K agonist 740YP partially reversed ajugol's effects, whereas PI3K inhibitor LY294002 mimicked them, verifying pathway involvement. In MSU-induced GA mice, oral ajugol administration alleviated joint swelling, reduced inflammatory cytokines and NLRP3 expression, preserved cartilage integrity, and restored autophagy via PI3K/AKT/mTOR suppression. Collectively, ajugol alleviates inflammation and cartilage damage by suppressing the PI3K/AKT/mTOR pathway, activating mitophagy, and inhibiting pyroptosis, providing a promising mitochondrial-targeted therapeutic strategy for gouty arthritis.
