Study of Retinoic Acid-Induced Osteoarthritis: Integrating RNA-sequencing, Network Pharmacology, Molecular Docking, and Experimental Validation

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.骨关节炎（Osteoarthritis, OA）是一种使人衰弱的关节疾病，以软骨退变与软骨细胞稳态失衡为核心特征。尽管视黄酸（Retinoic acid, RA）已被应用于骨关节炎的模型构建，但其具体作用靶点与信号通路仍未明确。本研究依托整合了RNA测序转录组学、网络药理学预测、计算配体-受体分子对接及生物学实验验证的转化研究框架，系统解析了RA在骨关节炎发病机制中的疾病修饰靶点及相关信号通路网络。对经RA处理的软骨细胞进行RNA测序分析，共筛选得到656个差异表达基因（differentially expressed genes, DEGs）。蛋白质相互作用（protein-protein interaction, PPI）网络分析与功能富集（GO/KEGG）分析揭示了多条关键通路，包括细胞外基质重塑、PI3K-Akt介导的机械转导等。分子对接结果显示，各靶点结合亲和力从高到低依次为：ACAN > SFRP2 > KANSL2（NSL2）等。网络药理学筛选得到42个RA与OA的共同靶点，其PPI分析与功能富集（GO/KEGG）结果涵盖肾素-血管紧张素系统、神经活性配体-受体相互作用等通路。分子对接总得分从高到低依次为：MAPK14(p38α)、PTGER3(PGE2-R)、CA2(CACNA2D2)等。本研究获取了RNA测序与网络药理学共有的5个交叉靶点：CA2、ACE、PTGS1(COX-1)、PGR及EDNRA(ETAR)，并通过分子对接将上述交叉靶点与RA进行对接，结果显示二者具有较强的结合亲和力；后续通过蛋白质印迹（western blot）与实时定量聚合酶链反应（RT-qPCR）实验进一步验证，证实RA可诱导上述交叉靶点的表达上调。此外，免疫荧光实验结果显示，MMP13水平升高且COL2A1表达受到抑制，共同反映了骨关节炎的表型特征。本研究确定CA2与ACE为多靶点调控的关键枢纽基因。本研究不仅揭示了RA干预骨关节炎的潜在作用机制，也为抗骨关节炎药物的研发提供了参考依据。