Cyasterone regulates lipid metabolism and autophagy mediated by the AMPK signaling pathway to improve KOA synovitis.

Abstract

BACKGROUND: Knee osteoarthritis (KOA) represents a chronic degenerative disorder associated with functional disability. Synovitis constitutes a major driver of KOA progression and clinical deterioration. Cyasterone (CYA) exerts therapeutic effects on KOA, yet its underlying mechanism remains undefined. PURPOSE: This study integrated non-targeted metabolomics with molecular biology approaches to investigate the therapeutic potential of CYA in KOA rats. METHODS: A KOA model was established by ACLT. After CYA intervention at graded concentrations, synovial tissue was subjected to pathological assessment and inflammatory evaluation. Non-targeted metabolomics was performed to characterize metabolic alterations. Fibroblast-like synovial cells (FLS) were isolated, and after group-specific treatment, analyses included EdU staining, Hoechst 33,342/PI double staining, flow cytometry, immunofluorescence, transmission electron microscopy (TEM), mRFP-GFP-LC3 puncta assay, Western blotting, and q-PCR. Molecular docking, molecular dynamics simulation (MDS), and surface plasmon resonance (SPR) techniques were explored the binding potential between CYA and the AMPK. RESULTS: Histopathological analysis demonstrated that CYA significantly alleviated synovitis progression in KOA. Metabolomics revealed extensive metabolic dysregulation in KOA, with CYA intervention restoring lipid metabolic balance. Molecular assays showed that CYA attenuated inflammatory cytokine production, suppressed FLS proliferation, and enhanced FLS apoptosis during KOA progression. Mechanistically, CYA regulated lipid metabolism in FLS through AMP-activated protein kinase (AMPK) activation, characterized by increased p-ACC and CPT1 expression, decreased FASN, SREBP-1, and SCD-1 expression, elevated GSH levels, reduced MDA accumulation, and diminished neutral lipid droplets. CYA also promoted AMPK-dependent autophagy, evidenced by upregulation of p-ULK1, LC3B, and Beclin1, concomitant downregulation of P62, enhanced autophagosome formation, and increased autophagic flux. Subsequent validation confirmed that modulation of lipid metabolism and autophagy via the AMPK pathway accounted for the protective effects of CYA against KOA synovitis. Molecular docking, MDS, and SPR results indicate that CYA exhibits high affinity for the AMPK protein. CONCLUSION: CYA activates AMPK signaling, restores lipid metabolic homeostasis, and enhances autophagy, thereby mitigating KOA synovitis.