Piperlongumine A attenuates RANKL-induced osteoclastogenesis by targeting MAPK signaling and downregulating c-Fos and NFATc1

Abstract

Bone homeostasis is tightly regulated by the equilibrium between osteoclasts and osteoblasts. Inhibiting osteoclast development with natural compounds presents a valuable approach to osteoporosis prevention and therapy. Piper longum L. (PL), traditionally employed in culinary and herbal medicine practices, has been reported to exhibit multiple bioactive properties, notably anti-inflammatory and antioxidant effects. However, its potential role in regulating bone metabolism remains largely unexplored. In this study, we isolated several compounds from PL extract (PLE) and evaluated their impact on osteoclast differentiation both in vitro and ex vivo. Using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR, we assessed the inhibitory effects of potential candidates on RANKL-induced osteoclast formation. Our results demonstrated that eight compounds exhibited significant inhibitory effects on RANKL-induced osteoclast differentiation in vitro, with piperlongumine A (PL-A) exhibiting the strongest activity. Moreover, PL-A suppressed RANKL-induced formation of TRAP-positive multinucleated osteoclasts in a dose-dependent manner and concurrently downregulated the mRNA expression of ATP6V0D2, CTSK, MMP-9, TRAP, and DC-STAMP. Additionally, PL-A significantly downregulated c-Fos and NFATc1, key regulators of osteoclastogenesis, in both RAW 264.7 cells and BMDMs stimulated with RANKL. These findings identify piperlongumine A (PL-A) as a potent inhibitor of RANKL-induced osteoclastogenesis, acting through downregulation of osteoclast-related genes and key transcription factors via the MAPK signaling pathway. PL-A shows strong potential as a natural therapeutic candidate for preventing bone loss and treating osteoclast-related diseases.