Hyperoside Suppresses Osteoclasts Differentiation and Function through Down-Regulating TRAF6/p38 MAPK Signaling Pathway

Abstract Background: Osteoporosis (OP) is a systemic metabolic bone disease that the bone resorption exceeds the bone formation,resulting in reduced bone mass, degeneration of the microstructure of bone tissue, and then increased bone fragility and fracture risk. Hyperoside (HP), as a natural product, can promote proliferation and differentiation of osteoblasts and presents a protective effect on ovariectomized (OVX) mice. However, the inhibitory effect of HP on osteoclasts (OCs) and the potential mechanism remains to be elucidated.Methods: In this study, RAW264.7 cells were used to generate OCs induced by RANKL, and HP was applied to the cell model. The effect of HP on OCs differentiation by TRAP-positive cell counting and TRAP activity test; Bone resorption assay and actin ring formation assay were used to verify the effect of HP on OCs function; The expression levels of osteoclast-specific genes and proteins were proved by RT-PCR and Western blotting.Results: HP significantly suppressed RANKL-induced OCs differentiation, function and the mRNAs expression levels of the osteoclast-related genes. Western Blotting results demonstrated that HP reduced the expression level of TNF receptor-associated factor 6 (TRAF6) and inhibited the p38 MAPK signing pathway by reducing the phosphorylation level of p38, which subsequently down-regulated the expression level of c-fos and NFATc1, ultimately, led to a decrease of the osteoclast-specific proteins CTSK and TRAP. In addition, TRAP-positive cell counting and TRAP activity test showed that HP could inhibit the differentiation of OCs; Bone resorption assay indicated that HP could restrain the OCs’ bone resorptive activity. Actin ring formation assay showed HP can cause the shrinkage of osteoclasts and disruption of actin ring structure.Conclusion: These results revealed that HP had an inhibitory effect on OCs by down-regulating TRAF6/p38 MAPK signaling pathway. Therefore, HP could be a promising natural compound for lytic bone diseases.