Glycyrrhizin improves bone metabolism in ovariectomized mice via inactivating NF-κB signaling

Abstract

Objectives

Postmenopausal osteoporosis (PMO) is a prevalent metabolic bone disease with high morbidity and serious complications. Here, we studied the effect of glycyrrhizin on bone metabolism using the ovariectomized (OVX) mouse model.

Methods

Osteoclast-related gene expression and osteoclastic function were evaluated in RAW264.7 cells and bone marrow-derived monocytes (BMMs) by real-time polymerase chain reaction and bone resorption assay. For animal studies, female C57BL/6J mice were randomly divided into sham operated, OVX and OVX with glycyrrhizin groups. Bone mass and trabecular microarchitecture were analyzed by micro-computed tomography, dual X-ray absorptiometry, and histomorphometric analysis. Receptor activator of nuclear factor-κB (NF-κB) ligand-induced osteoclastogenesis and the NF-κB signaling pathway were studied by tartrate-resistant acid phosphatase staining and western blotting, respectively.

Results

Glycyrrhizin inhibits RANKL-induced expression of Nfatc-1, c-Fos, Trap, Ds-stamp, and Ctsk in RAW264.7 cells. Also, fewer bone resorption pits form when BMMs are incubated in the presence of glycyrrhizin. Glycyrrhizin ameliorates bone loss and improves trabecular bone parameters in OVX mice. BMMs isolated from OVX mice show higher ability of RANKL-induced osteoclastogenesis, which is tremendously reversed by glycyrrhizin. There is significantly higher phosphorylation of IκB-α at Ser32 and NF-κB p65 at Ser536, as well as increased protein levels of c-FOS and NFATc-1 in BMMs of OVX mice, which are all greatly suppressed by glycyrrhizin.

Conclusions

Our findings imply that glycyrrhizin is a potential efficient adjuvant therapeutic for PMO.

摘要

目的：绝经后骨质疏松症（PMO）是一种常见的代谢性骨病, 发病率高, 并发症严重。本研究应用去卵巢（OVX）小鼠模型研究了甘草酸苷对骨代谢的影响。

方法：通过实时定量聚合酶链反应和骨吸收实验, 评估RAW264.7细胞和骨髓单核细胞（BMMs）中破骨细胞相关基因的表达和破骨功能。动物实验选用雌性C57BL/6J小鼠, 随机分为假手术组、去势组和去势+甘草酸苷组。通过显微计算机断层扫描、双X射线吸收测量仪和组织形态学分析法评估骨质量和骨小梁的微结构。分别通过抗酒石酸酸性磷酸酶染色和western blotting研究核因子-κB（NF-κB）配体诱导的破骨细胞生成的受体激活因子和NF-κB信号通路。

结果：甘草酸苷抑制RANKL诱导的RAW264.7细胞中Nfatc-1, c-Fos, Trap, Ds-stamp和Ctsk的表达。此外, 当骨髓基质细胞与甘草酸苷共同作用时, 形成的骨吸收陷窝较少。甘草酸苷改善了OVX小鼠的骨质流失并改善了骨小梁参数。从OVX小鼠中分离出的BMMs具有更高的RANKL诱导的破骨细胞生成的能力, 甘草酸苷可明显改善这一作用。在OVX小鼠的BMM中, Ser32的IκB-α和SER536的NF-κBp65的磷酸化水平显著升高, c-FOS和NFATc-1的蛋白水平明显升高, 甘草酸苷对以上指标均有明显的抑制作用。

结论：我们的研究结果提示甘草酸苷是一种潜在的有效的PMO辅助治疗药物。

Keywords:

• Ovariectomized mice
• postmenopausal osteoporosis
• glycyrrhizin
• NF-κB

Potential conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Additional information

Funding

This study was supported by National Natural Science Foundation for Youths [Grant No. 81701386], [Grant No. 81801397]; Zhejiang Provincial Administration of Traditional Chinese Medicine [Grant No. 2018ZA003]; Health Bureau of Zhejiang Province [Grant No. 2019KY007]; Zhejiang Provincial Natural Science Foundation [Grant No. LQ16H250001].