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

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.

Three Scrophularia Species (Scrophularia buergeriana, S. koraiensis, and S. takesimensis) Inhibit RANKL-Induced Osteoclast Differentiation in Bone Marrow-Derived Macrophages

Scrophulariae Radix, derived from the dried roots of Scrophularia ningpoensis Hemsl. or S. buergeriana Miq, is a traditional herbal medicine used in Asia to treat rheumatism, arthritis, and pharyngalgia. However, the effects of Scrophularia buergeriana, S. koraeinsis, and S. takesimensis on osteoclast formation and bone resorption remain unclear. In this study, we investigated the morphological characteristics and harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis, and compared the effects of ethanol extracts of these species using nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation. The harpagoside content of the three Scrophularia species was analyzed by high-performance liquid chromatography–mass spectrometry (HPLC/MS). Their therapeutic effects were evaluated by tartrate-resistant acid phosphatase (TRAP)-positive cell formation and bone resorption in bone marrow-derived macrophages (BMMs) harvested from ICR mice. We confirmed the presence of harpagoside in the Scrophularia species. The harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis was 1.94 ± 0.24 mg/g, 6.47 ± 0.02 mg/g, and 5.50 ± 0.02 mg/g, respectively. Treatment of BMMs with extracts of the three Scrophularia species inhibited TRAP-positive cell formation in a dose-dependent manner. The area of hydroxyapatite-absorbed osteoclasts was markedly decreased after treatment with the three Scrophularia species extracts. Our results indicated that the three species of the genus Scrophularia might exert preventive effects on bone disorders by inhibiting osteoclast differentiation and bone resorption, suggesting that these species may have medicinal and functional value.

SC-19220, a prostaglandin E2 antagonist, inhibits osteoclast formation by 1,25-dihydroxyvitamin D3 in cell cultures

1,25 Dihydroxy vitamin D3 (1,25(OH)2D3), prostaglandin (PG) E2 and parathyroid hormone (PTH) induce osteoclast formation in cell cultures. Previously, we have shown that SC-19220, an antagonist of the EP1 subtype of PGE receptors, inhibited tartrate-resistant acid phosphatase (TRAP)-positive cell formation by PGE2 and PTH in adherent cell cultures taken from neonatal rats. Since 1,25(OH)2D3 has been shown to induce osteoclast formation through PGE2 synthesis, in this study we have examined the effect of SC-19220 on osteoclast formation induced by 1,25(OH)2D3 in cell cultures by measuring bone resorption as well as TRAP-positive cell formation. SC-19220 inhibited osteoclast formation by 1,25(OH)2D3 as well as by PGE2 in cell cultures. The addition of SC-19220 to the later half but not to the earlier half of the culture inhibited 1,25(OH)2D3-induced formation. In the culture in which hydroxyurea was added in the later half period, SC-19220 inhibited osteoclast formation by 1, 25(OH)2D3. Under these conditions, 17-phenyl PGE1, an EP1 agonist, induced osteoclast formation. Thus, SC-19220 inhibits certain reactions in the later processes of osteoclast formation induced by 1,25(OH)2D3. In addition, SC-19220 also inhibited osteoclast formation induced by interleukin (IL)-11 and IL-6 as well as by PTH. It is suggested that the SC-19220 inhibiting reactions are shared by all the inducers including 1,25(OH)2D3 and are essential for osteoclast formation.