Detumescence Analgesic Plaster mitigates knee osteoarthritis via active ingredients targeting mitochondrial complex...

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.

This study aimed to systematically characterize DAP's chemical composition and decipher its chondroprotective pathways in KOA.

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.

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.

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.