Inhibitory Effect of Agrocybe Chaxingu (Chaxines) on RANKL-Induced Osteoclast Differentiation in RAW246.7 Cells: Possible Prevention of Osteoporosis

Abstract

Objectives: Receptor-activator of nuclear factor-κB ligand (RANKL) is an essential stimulating factor for inducing osteoclast differentiation, feasibly resulting in osteoporosis. The bioactive extract of Agrocybe chaxingu mushroom, CHX, has been shown to have osteoclastic inhibitory activity. Accordingly, we investigated if CHX would inhibit or disrupt RANKL-induced osteoclast differentiation, implying possible prevention of osteoporosis. Materials and Methods: Monocyte/macrophage RAW264.7 cells (RAW cells) were employed as our experimental model and treated with varying concentrations of RANKL alone or with CHX for 5 days. Formation of osteoclasts was then assessed using tartrateresistant acid phosphatase (TRAP) assay, counting stained cells as osteoclasts under a light microscope. The inhibitory mechanism of CHX was also explored by examining the RANKLmediated signaling pathways, oxidative stress (OXS), antioxidant enzymes, and apoptosis. Results: RANKL (100 ng/mL)-induced osteoclast differentiation in RAW cells was significantly (~20%) inhibited with 10 μg/mL of CHX. This was accompanied by the downregulation of two key signaling pathways and activation of antioxidant enzymes that likely led to the reduction in OXS. Moreover, CHX ultimately induced undifferentiated or RANKLunresponsive cells to apoptosis, indicated by the modulation of apoptotic regulators. These findings may then account for a disruption of osteoclast differentiation with CHX. Conclusion: CHX appears to have the inhibitory effect on RANKL-induced osteoclast differentiation in RAW cells. It is thus plausible that CHX may have potential clinical implications in osteoclast-mediated bone diseases/disorders, including osteoporosis.