Integrated analysis of bioinformatics, mendelian randomization, and experimental validation reveals novel diagnostic and therapeutic targets for osteoarthritis: progesterone as a potential therapeutic agent.

BACKGROUND: Osteoarthritis (OA), characterized by progressive degeneration of cartilage and reactive proliferation of subchondral bone, stands as a prevalent condition in orthopedic clinics. However, the precise mechanisms underlying OA pathogenesis remain inadequately explored. METHODS: In this study, Random Forest (RF), Least Absolute Shrinkage and Selection Operator (LASSO), and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) machine learning techniques were employed to identify hub genes. Based on these hub genes, an Artificial Neural Network (ANN) diagnostic model was constructed. The Drug Signatures Database (DSigDB) was utilized to screen small-molecule drugs targeting these hub genes, and molecular docking analyses and molecular dynamics simulations were employed to explore and validate the binding interactions between proteins and small-molecule drugs. Expression changes of the hub genes under inflammatory conditions were validated through in vitro experiments, including RT-qPCR and Western blotting, and the therapeutic effects of the identified small-molecule drug on chondrocytes under inflammatory conditions were further verified in vitro. Lastly, Mendelian randomization analysis was conducted to examine the causal association between progesterone levels and various OA phenotypes. RESULTS: In this study, we identified three hub genes: interleukin 1 receptor-associated kinase 3 (IRAK3), integrin subunit beta-like 1 (ITGBL1), and Ras homolog family member U (RHOU). An Artificial Neural Network (ANN) diagnostic model constructed based on these hub genes demonstrated excellent performance in both training and validation phases. Screening with the Drug Signatures Database (DSigDB) identified progesterone as a small-molecule drug targeting these key proteins. Molecular docking analysis using AutoDock Vina revealed that progesterone exhibited binding energies of ≤ -7 kcal/mol with each of the key proteins, indicating strong binding affinity. Furthermore, molecular dynamics simulations validated the stability and strength of these interactions. RT-qPCR and Western blotting confirmed the downregulation of the hub genes in IL-1β-treated chondrocytes. Western blotting also demonstrated the potential therapeutic effects of progesterone on IL-1β-treated chondrocytes. Finally, Mendelian randomization analysis established a significant association between progesterone levels and multiple OA phenotypes. CONCLUSION: In our study, IRAK3, ITGBL1, and RHOU were identified as potential novel diagnostic and therapeutic targets for OA. Progesterone was preliminarily validated as a small-molecule drug with potential effects on OA. Further research is crucial to elucidate the pathogenesis of OA and the specific therapeutic mechanisms involved.