More severe microdamage and micromechanical alterations: altered subchondral bone remodeling in varus knee osteoarthritis with osteoporosis.

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Abstract

[Indexed for MEDLINE] Conflict of interest statement: Declarations. Conflict of interest: None. 11. Phytomedicine. 2026 Feb;151:157789. doi: 10.1016/j.phymed.2026.157789. Epub 2026 Jan 7. Didymin halts knee osteoarthritis progression by targeting the GSK3B/5-LOX/11-HETE pathway to suppress chondrocyte ferroptosis. Zhou Y(1), Xue Y(1), Jiang M(1), Weiyang W(2), Chen W(3), Zhang J(4), Ji Y(1), Chen Z(1), Chen B(1), Lu H(5), Hu Y(1), Wan Z(1), He Q(2), Zhang H(1), Chen X(1), Fan Y(6), Wang H(7), Chen Z(6), Zhou C(8). Author information: (1)Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; The First Clinical of Medical School, Guangzhou University of Chinese Medicine, No. 12 Jichang Road, Baiyun District, Guangzhou, 510405, Guangdong Province, China; The Lab of Orthopaedics of Chinese Medicine of Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou, 510405, Guangdong Province, China. (2)Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China. (3)Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Guangdong Provincial Second Hospital of Traditional Chinese Medicine (Guangdong Provincial Engineering Technology Research Institute of Traditional Chinese Medicine), Guangzhou 510095, Guangdong Province, China. (4)Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; The Fifth Clinical College of Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China. (5)Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; The Third Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, 510240, Guangdong Province, China. (6)The Department of Orthopedics, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 16 Jichang Road, Baiyun District, Guangzhou, 510405, Guangdong Province, China. Electronic address: fanyinuo9557@126.com. (7)The Department of Orthopedics, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 16 Jichang Road, Baiyun District, Guangzhou, 510405, Guangdong Province, China. Electronic address: hipknee@163.com. (8)The Department of Orthopedics, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 16 Jichang Road, Baiyun District, Guangzhou, 510405, Guangdong Province, China; Maoming Hospital of Traditional Chinese Medicine, Maoming 525000, Guangdong Province, China. Electronic address: zcmzy1@163.com. BACKGROUND: Ferroptosis, an iron-dependent cell death mechanism, is a crucial factor in the progression of knee osteoarthritis (KOA), characterized by the excessive accumulation of lipid peroxides. Clinical and animal studies demonstrated increased iron deposition in KOA joints, primarily inside the subchondral bone marrow, synovium, and cartilage. Didymin, a flavonoid glycoside derived from Citrus species, demonstrates significant anti-inflammatory and antioxidant properties; however, its function in KOA remains unexplored. PURPOSE: Using metabolomics in conjunction with network pharmacology, molecular docking (MD), molecular dynamics simulation (MDS), surface plasmon resonance (SPR) assay, and in vivo experimental validation, this study sought to elucidate the therapeutic effects and potential mechanism by which the natural flavonoid Didymin mitigates KOA progression by targeting ferroptosis. MATERIALS AND METHODS: This study established an animal model of knee osteoarthritis (KOA). The rats received oral administration with Didymin at 0.7 mg/(kg·day) or 2.8 mg/(kg·day) weekly. The effects of Didymin on KOA cartilage and subchondral was evaluated using micro-CT, hematoxylin and eosin (H&E), safranin O/fast green (SO/FG), and toluidine blue (TB) staining, and immunohistochemistry (IHC). In vitro, CCK-8 assays and chondrocyte staining assessed cell proliferation and extracellular matrix metabolism to evaluate Didymin's effects on ferroptosis in primary chondrocytes induced by ferrous ammonium citrate and IL-1β. Subsequently, metabolomics was employed to identify differential metabolites, whilst integrating network pharmacology and disease gene target analysis to characterise protein targets in KOA and corresponding targets for Didymin. MD assessed interactions between Didymin and targets, with findings validated via MDS. In vitro SPR experiments directly validated these interactions. Downstream targets were further explored using protein-protein kinetic simulations and co-immunoprecipitation (Co-IP) techniques. Concurrently, immunofluorescence (IF), polymerase chain reaction (PCR), and Western blotting (WB) were employed to elucidate the mechanism by which Didymin ameliorates cartilage degeneration in knee osteoarthritis. RESULTS: In an iron-overload KOA rat model, Didymin treatment mitigated cartilage degradation and subchondral bone loss, improved ECM synthesis, and restored the expression of the antioxidant proteins nuclear factor E2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4). In vitro, Didymin suppressed ferroptosis in primary chondrocytes induced by ferric ammonium citrate and IL-1β. Metabolomics identified 11-hydroxyeicosatetraenoic acid (11-HETE) as a key differential metabolite in the KOA model. By combining metabolite-protein interaction networks, GSK3B is an upstream regulator of 11-HETE production. Further qPCR analysis of 5-LOX expression profiling identified it as a primary candidate. In vitro and in vivo experiments suggested that Didymin reduced the glycogen synthase kinase-3β (GSK3B) activation, resulting in the downregulation of 5-LOX expression and 11-HETE generation. The effect was validated using the GSK3B-specific inhibitor LY2090314, which mimicked Didymin's suppression of 5-LOX/11-HETE. Consequently, Didymin inhibited lipid peroxidation cascades by simultaneously suppressing 11-HETE generation and enhancing Nrf2/GPX4 activity. CONCLUSION: Our findings identify Didymin as a natural flavonoid compound that combats KOA through the GSK3B/5-LOX/11-HETE signaling pathway, offering a novel therapeutic strategy to alleviate ferroptosis-mediated cartilage destruction. Copyright © 2026 Elsevier GmbH. All rights reserved. DOI: 10.1016/j.phymed.2026.157789