Alphitolic acid suppressed osteoclast differentiation via hindering Cox-2/NF-κB signal pathway and alleviated titanium particle-induced bone loss in vivo.

Total joint arthroplasty (TJA) effectively improves the quality of life for patients with end-stage joint disorders. However, aseptic loosening caused by titanium (Ti) wear debris-induced osteolysis remains the leading cause of implant failure and revision surgeries. In this study, we investigated the therapeutic potential of alphitolic acid (ALA), a pentacyclic triterpenoid with known anti-inflammatory properties, against Ti particle-induced osteolysis and explored its underlying mechanisms. Network pharmacology analysis combined with molecular docking predicted that ALA targets key regulators of osteoclastogenesis, including PTGS2, with favorable binding affinity. In vitro, non-cytotoxic concentrations of ALA dose-dependently suppressed RANKL-induced osteoclast differentiation in BMMs, as evidenced by reduced TRAP-positive multinucleated cells and downregulation of osteoclast-specific genes. ALA also impaired F-actin ring formation and bone resorption activity. Mechanistically, ALA selectively inhibited phosphorylation of NF-κB p65 without affecting MAPK or AKT signaling pathways, leading to diminished expression and nuclear translocation of NFATc1 and c-Fos. RNA-sequencing further revealed that ALA downregulated Ptgs2 (COX-2) and its downstream targets, suggesting disruption of a COX-2/NF-κB feedback loop. In a murine calvarial osteolysis model, daily subcutaneous administration of ALA (15 mg/kg) significantly attenuated Ti particle-induced bone destruction, improving bone volume by 58% and reducing osteoclast numbers by 68% compared to vehicle-treated controls, as confirmed by micro-computed tomography and histomorphometric analysis. Collectively, these findings demonstrate that ALA effectively inhibits Ti particle-induced osteolysis by suppressing osteoclastogenesis through blockade of the NF-κB signaling pathway and modulation of PTGS2, positioning ALA as a promising natural compound for the prevention of periprosthetic osteolysis in TJA.