Inhibitory Effect of Bidens pilosa Extract on RANKL-Induced Osteoclast Differentiation of RAW264.7 Cells

Osteoclasts are associated with normal bone remodeling in healthy tissues and excess bone resorption in bone-destructive diseases. Bidens pilosa is a plant belonging to the Asteraceae family and is well known for its antioxidative stress effect. In the present study, RAW264.7 cells were treated with receptor activator of the NF-kappaB ligand (RANKL), which is predominant in the foci of pathological bone resorption, with or without extracts of B. pilosa harvested in Miyako Island, Japan (MBP) to examine the effects of MBP on osteoclastogenesis. Osteoclast differentiation was evaluated based on tartrate-resistant acid phosphatase (TRAP) activity. The expression of bone resorption-related enzymes and superoxide dismutase (SOD)-2 was examined using real-time polymerase chain reaction or enzyme-linked immunosorbent assays. The oxidative stress level was evaluated using fluorescent staining. Pit-formation assays with calcium phosphate disks were used to assess the mineral resorption activity. MBP suppressed the expression of carbonic anhydrase II, matrix metalloproteinase-9, and cathepsin K and decreased the size of TRAP-positive multinuclear osteoclast-like cells in the presence of RANKL. However, these small cells expressed bone resorption-related enzymes described above. MBP reduced the formation of large-sized resorption pits but not that of small-sized pits. Additionally, SOD-2 expression was increased, whereas RANKL-induced oxidative stress was decreased after the addition of MBP. In conclusion, MBP suppressed RANKL-induced osteoclastic bone resorption via an alleviation of oxidative stress. This inhibitory effect is thought to be exerted on the formation of large osteoclasts.