背景:EpimedinA(EA)已被证明可以抑制广泛的破骨细胞生成和骨吸收,但EA的影响仍未完全理解。我们研究的目的是研究EA对破骨细胞生成和骨吸收的影响,以探索相应的信号通路。
方法:大鼠随机分为假手术组和卵巢切除组,阳性对照组采用阿仑膦酸钠。通过测量骨密度和骨生物力学特性,系统分析了EA对骨质疏松症的治疗作用。体外,用核因子κB受体激活剂配体(RANKL)和巨噬细胞集落刺激因子(M-CSF)处理RAW264.7细胞以诱导破骨细胞分化。细胞活力测定,抗酒石酸酸性磷酸酶(TRAP)染色,和免疫荧光用于阐明EA对破骨细胞生成的影响。此外,使用蛋白质印迹分析或定量聚合酶链反应(PCR)评估骨分化相关蛋白或基因的表达,分别。
结果:口服EA干预3个月后,去卵巢大鼠显示骨密度增加,相对骨体积,小梁厚度,和小梁数量,以及减少小梁分离。EA剂量依赖性地使卵巢切除大鼠的骨密度和小梁微结构正常化。此外,EA抑制去卵巢大鼠TRAP和NFATc1的表达。此外,体外结果表明,EA通过抑制TRAF6/PI3K/AKT/NF-κB通路抑制破骨细胞分化。进一步的研究表明,对破骨细胞分化的影响,最初被EA抑制,当TRAF6基因过表达时被逆转。
结论:研究结果表明,EA可以通过抑制TRAF6/PI3K/AKT/NF-κB轴负向调节破骨细胞的生成,改善EA大鼠卵巢切除引起的骨质疏松症可能是一种有希望的治疗骨质疏松症的潜在策略。
BACKGROUND: Epimedin A (EA) has been shown to suppress extensive osteoclastogenesis and bone resorption, but the effects of EA remain incompletely understood. The aim of our study was to investigate the effects of EA on osteoclastogenesis and bone resorption to explore the corresponding signalling pathways.
METHODS: Rats were randomly assigned to the sham operation or ovariectomy group, and alendronate was used for the positive control group. The therapeutic effect of EA on osteoporosis was systematically analysed by measuring bone mineral density and bone biomechanical properties. In vitro, RAW264.7 cells were treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) to induce osteoclast differentiation. Cell viability assays, tartrate-resistant acid phosphatase (TRAP) staining, and immunofluorescence were used to elucidate the effects of EA on osteoclastogenesis. In addition, the expression of bone differentiation-related proteins or genes was evaluated using Western blot analysis or quantitative polymerase chain reaction (PCR), respectively.
RESULTS: After 3 months of oral EA intervention, ovariectomized rats exhibited increased bone density, relative bone volume, trabecular thickness, and trabecular number, as well as reduced trabecular separation. EA dose-dependently normalized bone density and trabecular microarchitecture in the ovariectomized rats. Additionally, EA inhibited the expression of TRAP and NFATc1 in the ovariectomized rats. Moreover, the in vitro results indicated that EA inhibits osteoclast differentiation by suppressing the TRAF6/PI3K/AKT/NF-κB pathway. Further studies revealed that the effect on osteoclast differentiation, which was originally inhibited by EA, was reversed when the TRAF6 gene was overexpressed.
CONCLUSIONS: The findings indicated that EA can negatively regulate osteoclastogenesis by inhibiting the TRAF6/PI3K/AKT/NF-κB axis and that ameliorating ovariectomy-induced osteoporosis in rats with EA may be a promising potential therapeutic strategy for the treatment of osteoporosis.