Inhibitory Effects of Rhaponticin on Osteoclast Formation and Resorption by Targeting RANKL-Induced NFATc1 and ROS Activity.

Jianbo He,Zhifeng Xiao,Jiewei Xie,Kunjing Zhong,Jinbo Yuan,Xiaojuan Li,Tiancheng Deng,Dingkun Lin,Kai Chen,Ziyi Wang,Delong Chen,Jiake Xu,Ronghe Gu

doi:10.3389/fphar.2021.645140

Abstract

The extravagant osteoclast formation and resorption is the main cause of osteoporosis. Inhibiting the hyperactive osteoclastic resorption is considered as an efficient treatment for osteoporosis. Rhaponticin (RH) is a small molecule that has been reported to possess anti-inflammatory, anti-allergic, anti-fibrotic, and anti-diabetic activities. However, the influence of RH on osteoclasts differentiation and function is still unclear. To this end, an array of assays including receptor activator of nuclear factor kappa-Β (NF-κB) ligand (RANKL) induced osteoclastogenesis, tartrate-resistant acidic phosphatase (TRAcP) staining, immunofluorescence, and hydroxyapatite resorption were performed in this study. It was found that RH had significant anti-catabolic effects by inhibiting osteoclastogenesis and bone resorption without cytotoxicity. Mechanistically, the expression of NADPH oxidase 1 (Nox1) was found to be suppressed and antioxidant enzymes including catalase, superoxide dismutase 2 (SOD-2), and heme oxygenase-1(HO-1) were enhanced following RH treatment, suggesting RH exhibited antioxidant activity by reducing the generation of reactive oxygen species (ROS) as well as enhancing the depletion of ROS. In addition, MAPKs, NF-κB, and intracellular Ca2+ oscillation pathways were significantly inhibited by RH. These changes led to the deactivation of osteoclast master transcriptional factor-nuclear factor of activated T cells 1 (NFATc1), as examined by qPCR and Western blot assay, which led to the decreased expression of downstream integrin β3, c-Fos, cathepsin K, and Atp6v0d2. These results suggested that RH could effectively suppress RANKL-regulated osteoclast formation and bone resorption. Therefore, we propose that RH can represent a novel natural small molecule for the treatment of osteoporosis by inhibiting excessive osteoclast activity.

Highlights

The well-orchestrated bone formation and bone resorption is vital for bone modelling and remodelling (Baron & Kneissel, 2013)
The primary antibodies special for nuclear factor of activated T cells 1 (NFATc1), c-Fos, cathepsin K (CTSK), V-ATPase-d2, integrin β3, extracellular signal regulated kinase (ERK), phosphorylated ERK (p-ERK), P38, phosphorylated P38 (p-P38), and β-actin were obtained from Cell Signaling Technology, Santa Cruz Biotechnology and Abcam companies
To determine the influence of RH on the formation progress of osteoclasts from bone marrow monocytes (BMMs) induced by RANKL, we performed an osteoclastogenesis assay as for the nuclei

Summary

Introduction

The well-orchestrated bone formation and bone resorption is vital for bone modelling and remodelling (Baron & Kneissel, 2013). Excessive bone resorption is considered to be the main cause of osteoporosis which is characterized by reduced bone mass, with the high risk of bone fractures (Rachner, Khosla, & Hofbauer, 2011). Estrogen deficiency and glucocorticoid administration are the top two primary causes of osteoporosis (Adami & Saag, 2019; Manolagas, O’Brien, & Almeida, 2013) and subsequent vertebral or hip fractures pose heavy economic burdens to patients and society (Melton, 2003). Available osteoporosis treatments such as hormone replacement therapy, bisphosphates, and RANKL antibody have acceptable therapeutic outcome by exhibiting an appreciated suppression on bone resorption. It is urgent to provide more effective alternative options to alleviate these osteoclast-related bone disorders

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Conclusion