Daidzein alleviates knee osteoarthritis via activation of the EGFR/Raf-1/ERK1/2 signaling pathway

BACKGROUND: Knee osteoarthritis (KOA) is a common degenerative joint disease marked by progressive articular cartilage loss, pathological subchondral bone remodeling, and inflammation-associated disruption of extracellular matrix (ECM) homeostasis. Disease-modifying options remain scarce, despite advances in symptomatic management. Daidzein (DAI), a soy-derived isoflavone, has demonstrated anti-inflammatory and chondroprotective effects; however, its key targets and downstream mechanisms in KOA remain unclear. OBJECTIVE: To investigate the hypothesis that DAI attenuates cartilage degeneration by activating the EGFR/Raf-1/ERK1/2 signaling pathway, thereby restoring ECM homeostasis. DESIGN: We used chondrocyte injury model and mouse model of KOA to systematically assess the chondroprotective effects of DAI. METHODS: Primary mouse chondrocytes were used to assess the effects of DAI on apoptosis, ECM homeostasis, and EGFR/Raf-1/ERK1/2 signaling under IL-1β-induced inflammatory conditions. Transcriptomic profiling was performed in control, model, and DAI-treated chondrocytes to provide direct evidence for target identification, followed by cross-analysis of reversely regulated genes, protein-protein interaction analysis, and topological prioritization. Molecular docking and molecular dynamics simulations were used to evaluate the structural basis and stability of the DAI-EGFR interaction. PROTAC-3 (GC19509), an EGFR-targeting PROTAC reagent, was used to probe the EGFR dependence of DAI actions. In vivo, mice subjected to destabilization of the medial meniscus (DMM) surgery received DAI by oral gavage (25 or 50 mg/kg/day), whereas celecoxib (31.43 mg/kg/day) served as a positive comparator. Cartilage degeneration, subchondral bone microarchitecture, inflammatory readouts, and EGFR/Raf-1/ERK1/2 signaling activity were then evaluated. RESULTS: Transcriptomic profiling identified EGFR as a DAI-responsive candidate target in IL-1β-injured chondrocytes. Cross-analysis of reversely regulated genes, together with protein-protein interaction and topological analyses, further prioritized EGFR as a hub target, whereas molecular docking and molecular dynamics simulations supported a stable interaction between DAI and EGFR. DAI significantly mitigated interleukin-1β (IL-1β)-induced chondrocyte injury, decreased apoptotic responses, and re-established ECM homeostasis, concomitant with upregulated EGFR/Raf-1/ERK1/ERK1/2 signaling. PROTAC-3 partly blunted DAI-driven EGFR/Raf-1/ERK1/2 activation and diminished the associated cytoprotective phenotype. In DMM-induced KOA mice, DAI reduced cartilage degeneration and corrected pathological subchondral bone remodeling, while lowering systemic inflammatory markers and reactivating EGFR/Raf-1/ERK1/2 signaling in cartilage. CONCLUSIONS: DAI activates EGFR/Raf-1/ERK1/2 signaling to protect chondrocytes from inflammation-related injury and maintain cartilage ECM homeostasis, thereby retarding KOA progression. These findings identify DAI as a promising disease-modifying candidate for KOA.