Osteoporosis (OP) is a common metabolic bone disease. Excessive osteoclastic activity significantly contributes to the development of OP. Icariin (ICA) is a flavonol glycoside derived from herbal plants and possesses curative effects on postmenopausal OP and bone fracture. This study aimed to investigate the effects of ICA on osteoclast differentiation induced by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and the involvement of estrogen receptor α (ERα) and RANK signaling cascade in this process. RANKL was used to induce the differentiation of RAW264.7 cells to into osteoclasts. Small interfering RNA technique was used to knockdown ERα in cells. Cell counting kit-8 assay was performed to determine the cytotoxicity of ICA. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was quantified by TRAP staining. RANKL induced the differentiation of RAW264.7 cells into osteoclasts, while ICA abolished the pro-osteoporotic effect of RANKL. Moreover, ERα knockdown abolished the effects of ICA on RANKL-induced osteoclastogenesis. Further exploration revealed that ICA inhibited the phosphorylation of c-Src in osteoclasts via regulating ERα, while inactivation of c-Src reversed ERα knockdown-promoted osteoclastogenesis. Lastly, ICA inhibited the activation of the mitogen-activated protein kinase signaling pathway and downregulated the expressions of target osteoclastogenic proteins in RANKL-treated RAW 264.7 cells, while ERα knockdown almost completely diminished the effects of ICA. ICA inhibited RANKL-induced osteoclast differentiation via regulating the ERα/c-Src/RANK signaling. These findings elucidated a novel mechanism by which ICA exerts an anti-osteoporotic effect.

中文翻译：

---

淫羊藿苷通过 ERα/c-Src/RANK 信号调节 RANKL 诱导的破骨细胞分化

骨质疏松症（OP）是一种常见的代谢性骨病。过度的破骨细胞活性显着促进 OP 的发生。淫羊藿苷（ICA）是一种从草本植物中提取的黄酮醇苷，对绝经后骨质疏松症和骨折具有疗效。本研究旨在探讨ICA对核因子κB受体激活剂(RANK)配体(RANKL)诱导的破骨细胞分化的影响以及雌激素受体的参与α（急诊室α）和RANK信号在此过程中级联。使用RANKL诱导RAW264.7细胞分化为破骨细胞。小干扰RNA技术用于敲低ERα在细胞中。进行细胞计数试剂盒8测定以确定ICA的细胞毒性。通过TRAP染色对抗酒石酸酸性磷酸酶(TRAP)阳性细胞的数量进行定量。 RANKL诱导RAW264.7细胞分化为破骨细胞，而ICA消除了RANKL的促骨质疏松作用。此外，急诊室α敲低消除了 ICA 对 RANKL 诱导的破骨细胞生成的影响。进一步的研究表明ICA抑制了磷酸化C-破骨细胞中的Src通过调节ERα，同时失活C-Src逆转ERα敲低促进破骨细胞生成。最后，ICA 抑制丝裂原激活蛋白激酶信号通路的激活，并下调 RANKL 处理的 RAW 264.7 细胞中目标破骨细胞蛋白的表达，而 ERα击倒几乎完全削弱了 ICA 的影响。 ICA 通过调节 ER 抑制 RANKL 诱导的破骨细胞分化α/C-Src/RANK 信令。这些发现阐明了 ICA 发挥抗骨质疏松作用的新机制。