Abstract
Osteoarthritis (OA) is related to over-proliferation or differentiation of osteoclasts. Although protocatechuic acid (PCA) has been identified to inhibit osteoclast differentiation and stimulate apoptosis in mature osteoclasts, whether it can relieve OA is still unknown. The present study aimed to investigate the effect of PCA on anterior cruciate ligament transection (ACLT)-induced OA and the potential mechanisms of action behind this effect. ACLT was performed on rats, which were then treated with or without PCA. C-terminal telopeptide of type I collagen (CTX-I) and CTX-II were tested in knee joint protein extracts by ELISA. Damage to cartilage was evaluated using Safranin-O/Fast Green staining. Osteoclast-related gene and protein expression was assessed through reverse transcription-quantitative PCR and western blotting. Tartrate-resistant acid phosphatase (TRAP) staining and functional bone resorption pit assays were performed using RAW264.7 murine macrophage cells to determine the effects of PCA on osteoclastic formation and function, respectively, in vitro. Finally, the activity of osteoclastogenesis-related signaling pathways was evaluated by western blotting. Levels of CTX-II were relatively decreased and Safranin-O/fast green staining indicated milder changes in the articular cartilage in the PCA treatment group. PCA downregulated osteoclast specific markers and suppressed receptor activator of nuclear factor-κB ligand-induced formation of TRAP-positive multinucleated cells, bone-resorption and pit formation. Mitogen-activated protein kinase (MAPK) and Akt signaling as well as the downstream factors, were downregulated by PCA. In conclusion, the present study demonstrated that PCA attenuated ACLT-induced OA by suppressing osteoclastogenesis by inhibiting the MAPK, ATK and NF-κB signaling pathways.