18β-Glycyrrhetinic Acid Inhibits Osteoclastogenesis In Vivo and In Vitro by Blocking RANKL-Mediated RANK-TRAF6 Interactions and NF-κB and MAPK Signaling Pathways.

Xiao Chen,Zili Qiu,Zhifeng Yin,Longjuan Qin,Xiaoqun Li,Jiacan Su,Xin Zhi

doi:10.3389/fphar.2018.00647

Abstract

Bone metabolism is determined by a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. The imbalance due to over-activated osteoclasts plays an important role in various diseases. Activation of NF-κB and MAPK signaling pathways by receptor activator of nuclear factor -κB ligand (RANKL) is vital for osteoclastogenesis. Here, we for the first time explored the effects of 18β-glycyrrhetinic acid (18β-GA), a pentacyclic triterpenoid found in the Glycyrrhiza glabra L roots, on RANKL-induced osteoclastogenesis, osteoclast functions and signaling pathways in vitro and in vivo. In bone marrow monocytes (BMMs) and RAW264.7 cells, 18β-GA inhibited osteoclastogenesis, decreased expression of TRAP, cathepsin K, CTR and MMP-9, blocked actin ring formation and compromised osteoclasts functions in a dose-dependent manner at an early stage with minimal effects on osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). For underlying molecular mechanisms, 18β-GA inhibited RANKL-induced phosphorylation of p65, p50, and IκB, blocked p65 nuclear translocation and decreased the DNA-binding activity of NF-κB. Besides, 18β-GA inhibited the activation of the MAPK pathways. Co-immunoprecipitation showed that 18β-GA treatment blocked RANK–TRAF6 association at an upstream site. In vivo, 18β-GA treatment inhibited ovariectomy-induced osteoclastogenesis and reduced bone loss in mice. Overall, our results demonstrated that 18β-GA inhibited RANKL-induced osteoclastogenesis by inhibiting RANK expression in preosteoclasts and blocking the binding of RANK and TRAF6 which lead to the inhibition of NF-κB and MAPK signaling pathways. 18β-GA is a promising novel candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

Highlights

Homeostasis of bone metabolism is determined by a dynamic balance between osteoclasts and osteoblasts (Chen et al, 2017b)
These results showed that 18β-glycyrrhetinic acid (18β-GA) had no significant effects on adipogenic differentiation and osteogenesis of bone marrow mesenchymal stem cells (BMSCs)
RT-polymerase chain reaction (PCR) showed that nuclear factor of activated T-cells 1 (NFATc1) was upregulated during osteoclastogenesis, while treatment with 18β-GA inhibited its upregulation (Figure 6C). These results showed that treatment with 18β-GA blocked the recruitment of TRAF6 after RANK was activated by RANKL and inhibited NFATc1 transcription by inhibiting the NF-κB and MAPK pathways (Figure 6D)

Summary

Introduction

Homeostasis of bone metabolism is determined by a dynamic balance between osteoclasts and osteoblasts (Chen et al, 2017b). Disruption of this balance with a higher rate of bone resorption over formation after osteoclast overactivation leads to pathological bone loss in various bone diseases including postmenopausal osteoporosis (PMOP) (Noel et al, 2017), rheumatoid arthritis (RA), and Paget’s disease. Inhibiting osteoclast overactivation is an important treatment strategy for pathological bone loss disease (Swart et al, 2016). The morbidity of osteoporosis correlates with age and sex (25.41% of females vs 15.33% of males), and PMOP is the most common form of osteoporosis (Majtan et al, 2017). There has been a significant increase in PMOP [14.94% (2005–2008) vs. 27.96% (2012–2015)] accompanied by a significantly increased morbidity among patients over 60 years of age (Chen et al, 2016; Jing et al, 2017)

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