Eugenol preserves chondrocyte extracellular matrix homeostasis by modulating the ALK1/ALK5-associated TGF-β/Smad signaling axis.

Osteoarthritis (OA) is characterized by inflammatory and catabolic disruption of cartilage extracellular matrix (ECM) homeostasis. An increased relative abundance of activin receptor-like kinase 1 (ALK1) relative to ALK5, together with altered Smad signaling, has been associated with chondrocyte dysfunction during OA progression. This study examined whether eugenol attenuates IL-1β-induced ECM dysregulation in primary human chondrocytes and whether ALK1/ALK5-associated Smad signaling contributes to this response. Primary human chondrocytes were treated to IL-1β in the presence or absence of eugenol and analyzed using RT-qPCR, Western blotting, immunofluorescence, exploratory RNA sequencing, and siRNA-mediated loss-of-function experiments. Receptor-expression profiles were also assessed in juvenile and aged mouse cartilage and in cartilage from a previously established ACLT cohort. Eugenol attenuated IL-1β-induced decreases in ACAN, COL2A1, and SOX9 expression and reduced IL-1β-induced increases in MMP13, ADAMTS5, COL10A1, RUNX2, and IL-6 immunoreactivity. At the protein level, eugenol increased COL2A1 abundance and reduced MMP13 and ADAMTS5 level. Exploratory pairwise RNA-seq analysis identified IL-1β-responsive genes that were directionally counter-regulated by both eugenol concentrations; these genes were enriched in inflammatory, cell-behavior, and TGF-β/BMP-related pathways. Prolonged IL-1β exposure increased the ALK1/ALK5 protein ratio, primarily because ALK5 declined more markedly than ALK1. Eugenol treatment was associated with a lower ALK1/ALK5 ratio, reduced Smad1/5/9 phosphorylation, and increased Smad2/3 phosphorylation. ALK1 knockdown phenocopied selected eugenol-associated ECM responses, whereas ALK5 knockdown weakened several matrix-restorative and anti-catabolic responses. Computational docking, short molecular-dynamics simulations, and Y279A/H280A mutagenesis provided exploratory observations but did not demonstrate direct eugenol-ALK1 binding. In mouse cartilage, receptor-expression profiles differed between juvenile and aged animals, and eugenol partially normalized ACLT-associated changes in ALK1 and ALK5 immunostaining. These findings indicate that eugenol attenuates IL-1β-induced ECM dysregulation in chondrocytes. Modulation of ALK1/ALK5-associated Smad signaling may contribute to this response; however, direct target engagement and ligand-specific signaling mechanisms remain to be established.