Gamabufotalin Inhibits Osteoclastgenesis and Counteracts Estrogen-Deficient Bone Loss in Mice by Suppressing RANKL-Induced NF-κB and ERK/MAPK Pathways.

Kaiqiang Sun,Le Huan,Jian Zhu,Jingchuan Sun,Fanqi Kong,Jiangang Shi,Changzhen Ren,Ximing Xu,Xiaofei Sun,Yi Deng

doi:10.3389/fphar.2021.629968

Abstract

Osteolytic bone disease is a condition of imbalanced bone homeostasis, characterized mainly by excessive bone-resorptive activity, which could predispose these populations, such as the old and postmenopausal women, to developing high risk of skeletal fragility and fracture. The nature of bone homeostasis is the coordination between the osteoblasts (OBs) and osteoclasts (OCs). Abnormal activation of osteoclasts (OCs) could compromise the bone homeostasis, constantly followed by a clutch of osteolytic diseases, including postmenopausal osteoporosis, osteoarthritis, and rheumatoid arthritis. Thus, it is imperatively urgent to explore effective medical interventions for patients. The traditional Chinese medicine (TCM) gamabufotalin (CS-6) is a newly identified natural product from Chansu and has been utilized for oncologic therapies owing to its good clinical efficacy with less adverse events. Previous study suggested that CS-6 could be a novel anti-osteoporotic agent. Nevertheless, whether CS-6 suppresses RANK-(receptor activator of nuclear factor-κ B ligand)/TRAF6 (TNF receptor-associated factor 6)-mediated downstream signaling activation in OCs, as well as the effects of CS-6 on OC differentiation in vivo, remains elusive. Therefore, in this present study, we aimed to explore the biological effects of CS-6 on osteoclastogenesis and RANKL-induced activation of related signaling pathways, and further to examine the potential therapeutic application in estrogen-deficient bone loss in the mice model. The results of in vitro experiment showed that CS-6 can inhibit RANKL-induced OC formation and the ability of bone resorption in a dose-dependent manner at both the early and late stages of osteoclastogenesis. The gene expression of OC-related key genes such as tartrate-resistant acid phosphatase (TRAP), CTSK, DC-STAMP, MMP9, and β3 integrin was evidently reduced. In addition, CS-6 could mitigate the systemic estrogen-dependent bone loss and pro-inframammary cytokines in mice in vivo. The molecular mechanism analysis suggested that CS-6 can suppress RANKL/TRAF6-induced early activation of NF-κB and ERK/MAPK signaling pathways, which consequently suppressed the transcription activity of c-Fos and NFATc1. Taken together, this present study provided ample evidence that CS-6 has the promise to become a therapeutic candidate in treating osteolytic conditions mediated by elevated OC formation and bone resorption.

Highlights

Bone tissue is highly dynamic, where bone remodeling occurs consistently via the formation of new bone and the elimination of old bone, and this homeostasis relies mainly on reciprocally delicate balance between osteoclast (OBs) and osteoclast (OCs) (Kim et al, 2018; He et al, 2017)
To investigate the cytotoxic effect of CS-6 on bone marrow–derived monocyte–macrophage (BMM), the cell viability was examined by CCK8 viability assay, and we found that CS-6 below 200 nM exhibited no dramatic damage to BMMs (Figures 1C–E)
Based on the results above, we explored the effect of CS-6 on the formation of functional OCs induced by RANKL in vitro

Summary

Introduction

Bone tissue is highly dynamic, where bone remodeling occurs consistently via the formation of new bone and the elimination of old bone, and this homeostasis relies mainly on reciprocally delicate balance between osteoclast (OBs) and osteoclast (OCs) (Kim et al, 2018; He et al, 2017). The bone remodeling is composed of three consecutive phases: OCs initiating bone resorption; catabolism conditioning the transition to anabolism condition; and OBs triggering bone formation. Each of these phases is under the control of the nervous system and endocrine system of the body (Negishi-Koga and Takayanagi, 2012). The MCSF ensures normal proliferation and survival of BMM cells via activating its receptor c-Fms and downstream Akt and ERK1/2 signaling pathways, while RANKL is indispensable for promoting the differentiation of OCs by its receptor RANK which could interact with TNF receptor-associated factor 6 (TRAF6) and activate a series of downstream cascades, including mitogenactivated protein kinases (MAPK) and NF-κB pathways (Biskobing et al, 1995; Zhou et al, 2016; Kim et al, 2017). Agents that could target those key transcription factors or genes may ameliorate the excessive activated OCs and facilitate the treatment of bone loss–related diseases

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