BACKGROUND: Osteoarthritis (OA) stands as a widespread joint condition that involves cartilage degeneration, meniscal injury, and synovial lesions, imposing a substantial economic and societal burden. Homoplantaginin (Homo), a flavonoid glycoside, is the primary active constituent of the traditional herbal medicine Salvia plebeia R.Br., and exhibits notable anti-inflammatory and anti-oxidative properties. Nevertheless, its therapeutic potential for OA remains largely unexplored.
PURPOSE: This study aimed to evaluate the therapeutic efficacy of Homo in the treatment of OA and to elucidate its underlying mechanisms.
STUDY DESIGN: By integrating in vivo and in vitro OA models, with transcriptomics analysis, molecular-protein affinity analysis, we identified the protective pathway implicated in the therapeutic mechanism of Homo against OA.
METHODS: The therapeutic effect of Homo on OA was assessed using the anterior cruciate ligament transection (ACLT) mouse model in vivo and the LPS-induced chondrocytes model in vitro. Micro-CT and histological analysis were employed to evaluate the Homo's protective impacts on articular cartilage or subchondral bone in vivo. In vitro, CCK-8 assays, apoptosis analysis, Western blot, Real-time quantitative PCR (RT-qPCR), reactive oxygen species (ROS) staining assessed the Homo's influence on chondrocytes viability, oxidative stress, and extracellular matrix (ECM) metabolism. Subsequently, transcriptomics analysis, molecular docking, molecular dynamics simulations, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) were performed to identify the potential therapeutic targets of Homo in OA. Furthermore, Western blot, RT-qPCR, immunofluorescence (IF), JC-1 staining, and Transmission electron microscope (TEM) were utilized to explore the modulation of Homo in mitochondrial function and mitophagy-related signaling pathways in chondrocytes.
RESULTS: Homo significantly alleviated OA-related pathological manifestations in both in vivo and in vitro. These beneficial effects were evidenced by reduced cartilage degradation, normalized ECM metabolism, enhanced chondrocytes viability, suppression of apoptosis, and oxidative stress. Transcriptomics sequencing revealed mitophagy as a potential mechanism underpinning Homo's therapeutic action, accompanied by a marked upregulation of Sirt3 expression. Sirt3 was subsequently identified as a direct target protein of Homo via combined molecular docking, molecular dynamics simulations, CETSA, and SPR analysis. Further investigation demonstrated that Homo activates the Sirt3/PINK1/Parkin signaling pathway, thereby promoting mitophagy and restoring mitochondrial function. Ultimately, the therapeutic efficacy of Homo in ameliorating OA via targeting Sirt3 was conclusively validated through rescue experiments conducted in vivo and in vitro.
CONCLUSION: This research elucidates that Homo ameliorates chondrocytes homeostasis and attenuates OA progression for the first time. We demonstrate that Homo directly binds to Sirt3, thereby activating the downstream PINK1/Parkin signaling axis and enhancing mitophagy. This finding provides an innovative therapeutic strategy for OA clinical management.
