Detumescence Analgesic Plaster mitigates knee osteoarthritis via active ingredients targeting mitochondrial complex 1/AMPK/MYL3-regulated cartilage homeostasis

BACKGROUND: The Detumescence Analgesic Plaster (DAP) has been widely used in clinical practice for knee osteoarthritis (KOA) treatment, yet its active ingredients and molecular mechanisms remain incompletely understood. PURPOSE: This study aimed to systematically characterize DAP's chemical composition and decipher its chondroprotective pathways in KOA. METHODS: A papain-induced KOA rat model was employed to evaluate DAP's therapeutic effects through behavioral assessments (mechanical withdrawal threshold, gait analysis) and histological evaluations (H&E, safranin O-fast green staining). UPLC-Q-TOF/MS combined with Franz diffusion cells identified DAP's chemical profile. RNA-seq was performed to compare gene expression between KOA and DAP-treated groups, followed by protein-protein interaction (PPI) and gene co-expression network analysis to prioritize key targets. Validation was conducted using Western blot, qPCR, and immunohistochemistry. IL-1β-stimulated chondrocytes were used to screen active ingredients and validate their effects on mitochondrial function. RESULTS: DAP treatment significantly alleviated pain, restored joint mobility, and preserved cartilage integrity in KOA rats. Chemical profiling identified 92 compounds, including 28 active ingredients with high transdermal permeability. RNA-seq revealed 206 DAP-reversed genes primarily associated with mitochondrial dysfunction, oxidative stress, and inflammatory signaling. Network analysis pinpointed 23 core targets, with mitochondrial complex I subunits (NDUFA5, NDUFA6, NDUFS6), AMPK, and MYL3 emerging as critical nodes in oxidative phosphorylation. DAP restored the expression of these targets in KOA cartilage. In vitro experiments demonstrated that 1,5-dicaffeoylquinic acid, verproside, and catalposide attenuated ROS production, enhanced ATP synthesis, and stabilized mitochondrial membrane potential via the NDUFA6/AMPK/MYL3 axis, thereby inhibiting chondrocyte apoptosis. CONCLUSION: This study provides the first evidence that DAP exerts chondroprotective effects by ameliorating mitochondrial dysfunction and oxidative stress in KOA through the mitochondrial complex I/AMPK/MYL3 signaling pathway. These findings offer a mechanistic basis for DAP's clinical efficacy and highlight potential therapeutic targets for KOA management.