Estrogenic activity and mechanism of action of puerarin phytoestrogen on mouse osteoclast

Abstract

The effects and mechanisms of action of Pueraria mirifica extract (PME) and puerarin (PU) on primary mouse osteoclasts and cross-talk between osteoblasts and osteoclasts were investigated. Three experimental series were set up. Experiment I: to study the effects of PME and PU on anti-osteoclastogenesis, mouse bone marrow macrophages (BMMs) were incubated with 0.3% DMSO, 1, 5 and 10 mg/ml PME, 0.1, 10 and 1000 nM PU, and 10 nM E2 for 24 h, and cell proliferation, differentiation, fusion and resorptive function were determined. PME and PU suppressed the RANKL-induced differentiation of BMMs into pre-osteoclasts and the fusion of pre-osteoclasts to multi-nucleated mature osteoclasts. The PME showed a differential dose-response effect, where a low PME dose (1 mg/ml) suppressed the fusion stage of mononuclear pre-osteoclast and a high PME dose (10 mg/ml) suppressed the differentiation from BMMs to pre-osteoclasts and decreased mRNA expression levels of Nfatc1 and Tm7sf4. The mechanisms of the low PME dose and PU were passed through estrogen receptors (ERs). Nevertheless, the anti-osteoclastogenic activity of a high PME dose was not significantly suppressed by ER antagonists, suggesting that PME might act via one or more alternative pathway(s). Experiment II: to study the effects of PME and PU on induction of osteoclast apoptosis, BMMs were treated with 0.3% DMSO, 10 nM E2, 1 and 10 mg/ml PME and 1000 nM PU for 72h, and cell apoptosis was determined. PME, but not PU and E2, induced apoptosis in mouse BMMs-preosteoclasts, while FasL mRNA expression levels were decreased after 24-h exposure to all three treatments. Experiment III: to study the effects of PME and PU on the cross-talk between osteoblasts and osteoclasts, each single-cell type culture (either mouse osteoblast ST2 precursors or BMMs) and a two-cell type coculture condition were conducted. Cells were treated with 0.025% DMSO, 10 nM E2, 1 mg/ml PME and 1000 nM PU. Exposure of ST2 cells alone with the PME reduced Rankl expression, which led to a significant decrease in Rankl/Opg ratio. However, exposure of BMMs alone or two-cell type coculture with PME, PU and E2 had no effects on expression of Rankl, Opg, Rankl/Opg ratio, Rank, EphB4 and EfnB2. In conclusion, PME and PU elicit the anti-osteoclastogenic activity of both directly on osteoclasts and indirectly via the regulation of osteoblasts through RANKL/OPG/RANK signal This study increases the knowledge and understanding of the mode of action of PME and PU on bone cells which corroborates the high potential of PME and PU to be developed as anti-resorptive agents for osteoporotic patients.