Objective

Given the limitations of current anti-resorption agents for postmenopausal osteoporosis, there is a need for alternatives without impairing coupling crosstalk between bone resorption and bone formation ie. osteoclastogenesis. Puerarin, a unique C-glycoside isoflavonoid, was found to be able to prevent bone loss by inhibiting bone resorption, but the underlying mechanism was controversial. In this study, we investigated the effects of puerarin on osteoclastic differentiation, activation and bone resorption and its underlying molecular mechanism in vitro, and then evaluated the effects of puerarin on bone metabolism using an ovariectomized (OVX) rat model.

Methods

In vitro, the effect of puerarin on osteoclastic cytotoxicity, differentiation, apoptosis, activation and function were studied in raw 264.7 ​cells and mouse BMMs. Mechanistically, osteoclast-related makers were determined by RT-PCR, western blot, immunofluorescence, and kinase activity assay. In vivo, Micro-CT, histology, serum bone biomarker, and mechanical testing were used to evaluate the effects of puerarin on preventing osteoporosis.

Results

Puerarin significantly inhibited osteoclast activation and bone resorption, without affecting osteoclastogenesis or apoptosis. In terms of mechanism, the expressions of protein of integrin-β3 and phosphorylations of Src, Pyk2 and Cbl were lower in puerarin group than those in the control group. Oral administration of puerarin prevented OVX-induced trabecular bone loss and significantly improved bone strength in rats. Moreover, puerarin significantly decreased trap positive osteoclast numbers and serum TRAP-5b, CTx1, without affecting bone formation rate.

Conclusions

Collectively, puerarin prevented the bone loss in OVX rat through suppression of osteoclast activation and bone resorption, by inhibiting integrin-β3-Pyk2/Cbl/Src signaling pathway, without affecting osteoclasts formation or apoptosis.

Translational potential of this article

These results demonstrate the unique mechanism of puerarin on bone metabolism and provide a novel agent for prevention of postmenopausal osteoporosis.

中文翻译：

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葛根素通过阻断整合素-β3 Pyk2/Src/Cbl信号通路特异性破坏破骨细胞活化

客观的

鉴于目前用于绝经后骨质疏松症的抗吸收剂的局限性，需要在不损害骨吸收和骨形成之间的耦合串扰的情况下使用替代物，即。破骨细胞生成。葛根素是一种独特的 C-糖苷类异黄酮，被发现能够通过抑制骨吸收来预防骨质流失，但其潜在机制存在争议。在这项研究中，我们在体外研究了葛根素对破骨细胞分化、活化和骨吸收的影响及其潜在的分子机制，然后使用去卵巢（OVX）大鼠模型评估了葛根素对骨代谢的影响。

方法

在体外，在原始 264.7 细胞和小鼠 BMM 中研究了葛根素对破骨细胞的细胞毒性、分化、凋亡、活化和功能的影响。机制上，破骨细胞相关标志物通过 RT-PCR、蛋白质印迹、免疫荧光和激酶活性测定来确定。体内、Micro-CT、组织学、血清骨生物标志物和力学测试用于评估葛根素预防骨质疏松症的作用。

结果

葛根素显着抑制破骨细胞活化和骨吸收，而不影响破骨细胞生成或细胞凋亡。从作用机制上看，葛根素组整合素-β3蛋白表达及Src、Pyk2、Cbl磷酸化水平低于对照组。葛根素的口服给药可防止 OVX 诱导的骨小梁丢失并显着改善大鼠的骨强度。此外，葛根素显着降低了trap阳性破骨细胞数量和血清TRAP-5b、CTx1，而不影响骨形成率。

结论

葛根素通过抑制整合素-β3-Pyk2/Cbl/Src 信号通路抑制破骨细胞活化和骨吸收来防止 OVX 大鼠的骨丢失，而不影响破骨细胞的形成或细胞凋亡。

本文的转化潜力

这些结果证明了葛根素对骨代谢的独特机制，为预防绝经后骨质疏松症提供了一种新的药物。