Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-\u03baB pathway

Supatta Chawalitpong,Soichiro Nakamura,Shigeru Katayama,Patcharee Ritprajak,Narattaphol Charoenphandhu,Ratchanaporn Chokchaisiri,Takakazu Mitani,Asada Leelahavanichkul,Apichart Suksamrarn,Tanapat Palaga,Ratchaneevan Aeimlapa,Ahmad Ai Athamneh

doi:10.1038/s41598-018-23912-3

Abstract

Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC50) for osteoclast differentiation was 36.69 μM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

Highlights

Osteoporosis is one of the major health concerns for aging communities
We reported that cyperenoic acid has strong anti-osteoclastogenic activity in an in vitro model of RANKL-induced bone marrow (BM)-derived osteoclast differentiation
To determine the toxicity of cyperenoic acid on macrophage-like cell line RAW264.7 or BMs-derived osteoclast precursors, cells were treated with cyperenoic acid at various concentrations (0–100 μM) and the cell viability was measured

Summary

Introduction

Osteoporosis is one of the major health concerns for aging communities. There are two types of osteoporosis which are postmenopausal osteoporosis occurred in woman after menopause and senile osteoporosis occurred in both men and women often over 70 years of age[1]. The costimulatory signaling via PLCγ-Ca2+ is crucial for osteoclast differentiation program[8] These downstream early intracellular signaling culminates in activation of key transcription factors in osteoclastogenesis, including c-Fos, AP-1 and NFATc12. Among these transcription factors, NFATc1 plays essential roles in osteoclast differentiation[9]. Senescence Accelerated Mouse Prone 6 (SAMP6) developed by Takada et al (1981)[14] from AKR/J strains is a useful mouse model of senile osteoporosis. They are characterized by a low peak in bone mass early on after the age of 16 weeks[15]. Until now there is no report on the effect of cyperenoic acid on osteoclastogenesis

Methods

Results

Conclusion