Abstract: BACKGROUND: Metformin is currently considered the first-line medication for the treatment of type 2 diabetes. Metformin may delay the progression of osteoarthritis, but its specific mechanism of action remains unclear.
OBJECTIVE: To evaluate the therapeutic effects and the related action mechanisms of metformin on osteoarthritis in rats.
METHODS: (1) Network pharmacology: Potential common targets for metformin, osteoarthritis, and ferroptosis were screened using the CTD, SwissTargetPrediction, GeneCards, and OMIM databases. After importing the targets into the STRING database, protein-protein interaction analysis was conducted to identify the key targets for metformin, osteoarthritis, and ferroptosis. (2) Molecular docking: P53 and its downstream factor SLC7A11 protein structures in PDB format were downloaded from the PDB database. The 2D structure of metformin was converted to a 3D structure, and molecular docking of metformin with the proteins was performed using Discovery Studio 2019 Client. (3) In vivo experiments: Thirty male SD rats were randomly divided into three groups (n=10). The blank group did not receive surgery. The osteoarthritis model was established using the modified Hulth method for the model and metformin groups. One day after the surgery, rats in the metformin group were gavaged with metformin 200 mg/kg per day, while the blank and model groups were gavaged with physiological saline. Treatment continued for 4 weeks. Hematoxylin-eosin staining and Safranin O-fast green staining were used to observe the pathological morphology and structure of the knee cartilage, and Mankin scoring was performed. ELISA was used to measure the levels of tumor necrosis factor-α and interleukin-6 in the serum. The microplate method was used to measure serum ferroptosis-related indicators, including glutathione, malondialdehyde, and Fe2+. Immunofluorescence staining, western blot assay, and real-time qPCR were used to detect the protein and mRNA expression of P53, SLC7A11, glutathione peroxidase 4, proteoglycans, and matrix metalloproteinase 13 in the cartilage tissue of the rats.
RESULTS AND CONCLUSION: (1) A total of 96 intersecting targets among metformin, osteoarthritis, and ferroptosis were identified. After protein-protein interaction analysis, 77 potential targets were found. Further screening identified the core targets as TP53, AKT1, JUN, interleukin-6, MYC, interleukin-1β, and tumor necrosis factor-α, among others. (2) Docking analysis results showed that metformin bound strongly and stably with P53 and its downstream factor SLC7A11. (3) In the model group, the knee cartilage surface was irregular, with cartilage tissue defects and reduced chondrocyte numbers. Compared to the model group, the knee cartilage structure damage in the metformin group was significantly improved, with a smoother cartilage surface and increased chondrocyte numbers. The Mankin score in the model group was significantly higher than that in the blank group, while the Mankin score in the intervention group was significantly lower than that in the model group. (4) Compared with the model group, the metformin group had significantly lower levels of tumor necrosis factor-α, interleukin-6, malondialdehyde, and Fe2+, and significantly higher glutathione levels. (5) Compared to the model group, the metformin group had significantly increased protein and mRNA expression of SLC7A11, glutathione peroxidase 4, and proteoglycans, and significantly decreased protein and mRNA expression of P53 and matrix metalloproteinase 13 in their cartilage tissue. (6) The results indicate that metformin can effectively improve cartilage damage in osteoarthritis rats and alleviate chondrocyte ferroptosis by inhibiting the aberrantly activated P53/SLC7A11/glutathione peroxidase 4 signaling pathway. This improvement in chondrocyte iron metabolism and lipid peroxidation response further reduces cartilage matrix degradation and prevents further cartilage damage and inflammatory response.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: osteoarthritis, articular cartilage, ferroptosis, iron metabolism, metformin, P53, SLC7A11, glutathione peroxidase 4, engineered tissue construction