Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis

Huey-En Tzeng,Huey-En Tzeng,Po-Hao Huang,Yi-Ying Wu,Tzu-Hung Lin,Ying-Ming Chiu,Chun-Hao Tsai,Chun-Hao Tsai,Tsurng-Juhn Huang,Yi-Ju Lee,Gregory J Tsay,Gregory J Tsay,Ying-Ming Chiu

doi:10.1038/s41598-018-29257-1

Abstract

NC-8 (ent-16-oxobeyeran-19-N-methylureido) is an isosteviol-derived analogue with multiple biological effects, including anti-inflammation and anti-bacterial activities and inhibition of HBV viral surface antigen gene expression. In this study, we explored the effects of NC-8 on the formation of osteoclasts from RAW 264.7 cells. We found that NC-8 exerts the novel effect of inhibiting osteoclast-like cell formation. Our experiments showed that RANKL-induced ERK, p38, and JNK phosphorylation were inhibited by NC-8. An ovariectomy-induced osteoporosis animal model was used to examine the protective effects of oral treatment with NC-8. Serum analysis was used to examine markers of osteoblasts, osteoclasts, and renal and hepatic function in rats. Micro CT scanning and histological analysis were used to measure bone loss in ovariectomized rats. Oral administration of NC-8 effectively decreased excess bone resorption and significantly antagonized trabecular bone loss in ovariectomized rats. Serum analysis of C-terminal telopeptide of type-I collagen, an osteoclast marker, also showed that NC-8 administration inhibited excess bone resorption. Furthermore, serum analysis showed that renal and liver function were not affected by these doses of NC-8 during long-term treatment. Our results demonstrate that NC-8 inhibits osteoclast differentiation and effectively ameliorates ovariectomy-induced osteoporosis.

Highlights

The molecular weight of NC-8 is 346.51. (B) Cell viability tests were performed by MTT detection
RAW 264.7 cells were treated with increasing concentrations (0, 1, 5, 10, 20, 40, and 80 μg/ml) of NC-8 for 72 h, and cell viability was analyzed using the MTT assay
The results showed that incubation with 100 ng/ml RANKL and 10 μg/ml NC-8 reduced RANKL-induced osteoclast differentiation

Summary

Introduction

The molecular weight of NC-8 is 346.51. (B) Cell viability tests were performed by MTT detection. After 3, 6, 9, and 12 days, MTT reagents were added to each well, and the absorbances were measured at 550 nm; the cell viability was calculated. Isosteviol derivatives exert a variety of biological effects, including anti-hypertension, anti-inflammation, anti-diarrhea, anti-cancer, anti-viral, and anti-bacterial activity[12,13,14,15,16]. NC-8, an isosteviol derivative, has been demonstrated to effectively inhibit the secretion of hepatitis B virus (HBV) surface antigens and HBV core antigens and inhibit the host TLR2/NF-κB signaling pathway[14]. The effect of NC-8 on RANKL activity through the ERK, p38, and NF-κB pathways has not been identified in the existing literature. We investigated how the isosteviol-derived novel compound NC-8 affects the differentiation of osteoclasts

Methods

Results

Conclusion