Eupatorin as a Promising Natural Compound Against Knee Osteoarthritis: From Network Pharmacology to Experimental...

Knee osteoarthritis (KOA), a chronic degenerative joint disease, is primarily driven by inflammation-induced cartilage degradation, which represents its core pathological feature. Eupatorin, with its distinct anti-inflammatory properties, has emerged as a promising candidate for KOA research. This study aimed to explore the therapeutic potential of Eupatorin and elucidate its underlying mechanisms in KOA through an integration of network pharmacology analysis and experimental validation.

Potential targets of Eupatorin and KOA-related genes were retrieved from multiple databases, and the overlapping targets were utilized to build a protein‒protein interaction (PPI) network to identify core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to characterize the associated biological processes (BP), molecular functions (MF), and cellular components (CC). Additionally, molecular docking was performed to assess the binding affinities of Eupatorin with the core targets. Direct target engagement was confirmed using a cellular thermal shift assay (CETSA). Finally, biological experiments using interleukin-1β (IL-1β)-stimulated primary rat chondrocytes were carried out to validate the protective effects of Eupatorin through its anti-inflammatory activity.

Network pharmacology analysis revealed 46 overlapping targets, with Matrix Metallopeptidase 9 (MMP9), Epidermal Growth Factor Receptor (EGFR), and Prostaglandin G/H synthase 2 (PTGS2) as key nodes within the PPI network. GO and KEGG enrichment analyses revealed significant associations with inflammatory responses and extracellular matrix (ECM) metabolism, particularly the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and estrogen signalling pathways. Molecular docking further confirmed strong binding affinities between Eupatorin and key targets, including MMP9, EGFR, and PTGS2. CETSA validated the direct binding of Eupatorin to PTGS2. Eupatorin significantly inhibited IL-1β-induced cytokine expression and ECM degradation while promoting ECM synthesis and restoring impaired autophagy in inflamed chondrocytes, as indicated; however, no significant effect on cellular senescence was observed. Mechanistically, Eupatorin exerted its protective effects on chondrocytes by attenuating the upregulation of the PI3K/AKT and estrogen signalling pathways.

Eupatorin has demonstrated potential for use in KOA therapy by targeting inflammation and ECM, and by regulating the PI3K/AKT and estrogen-associated signaling pathways.