ABSTRACT: Osteoarthritis (OA) is a debilitating joint disorder characterized by cartilage degradation and chondrocyte homeostasis disruption. Although retinoic acid (RA) has been applied in the model preparation of OA, its target of action and signaling pathway are not clear. Leveraging a translational framework that integrates RNA-sequencing transcriptomics, network pharmacology prediction, computational ligand-receptor molecular docking, and biological experimental validation, this study systematically deciphers RA's disease-modifying targets and associated signaling circuitry in OA pathogenesis. RNA-sequencing of RA-treated chondrocytes revealed 656 differentially expressed genes (DEGs). Protein-protein interaction (PPI) network analysis and functional enrichment (GO/KEGG) highlighted key pathways, including extracellular matrix reorganization and PI3K-Akt-mediated mechanotransduction and others. Additionally, molecular docking results ranked binding affinities in descending order as follows: ACAN > SFRP2 > KANSL2 (NSL2), and so on. Network pharmacology identified 42 RA-OA shared targets, with protein-protein interaction (PPI) analysis and functionally enriched (GO/KEGG) including the Renin-angiotensin system, Neuroactive ligand-receptor interaction, and others. The molecular docking results showed a total score in descending order of MAPK14(p38α), PTGER3(PGE2-R), CA2(CACNA2D2), and so on. Five intersecting targets (CA2, ACE, PTGS1(COX-1), PGR, and EDNRA(ETAR) ) from RNA-sequencing and network pharmacology were obtained, and molecular docking was used to dock the intersecting targets to RA. The docking results demonstrated strong binding affinities, which were further validated through western blot and RT-qPCR experiments, confirming the RA-induced upregulation of the intersecting targets. These findings were accompanied by immunofluorescent evidence showing elevated levels of MMP13 and suppressed expression of COL2A1, collectively reflecting the phenotypic characteristics of OA. Our findings position CA2 and ACE as key hubs for multi-targeting. This study not only reveals possible mechanistic studies of RA in OA but also provides a drug reference for the development of drugs against OA.
