"Betaone" barley water extract suppresses ovariectomy-induced osteoporosis in vivo and RANKL-induced osteoclast differentiation in vitro

Abstract

Betaone is a variety of barley developed by the Korea Rural Development Administration. This study investigated the anti-osteoporosis effects of Betaone barley water extract (B1W) on ovariectomy (OVX)-induced bone loss in mice. To elucidate its mechanism, the effect of B1W on osteoclasts was assessed by measuring the protein expression of nuclear factor-activated T cells c1 (NFATc1), the expression of genes involved in osteoclast differentiation, and bone pit assays. B1W (300 mg/kg/day) significantly increased bone mineral density and bone volume fraction, but decreased trabecular separation compared to the OVX group. B1W also showed a trend towards decreasing serum C-telopeptide of collagen type 1 levels in OVX mice. Additionally, B1W reduced the expression of NFATc1 and downregulated the mRNA expression levels of various marker genes such as c-Fos, tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated Ig-like receptor (OSCAR). B1W reduced the osteoclast activity in the receptor activator of nuclear factor-κB ligand (RANKL)-treated osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Based on the results, B1W can be considered a useful candidate for a therapeutic agent for treating conditions of bone loss and could also be used as an ingredient in health supplements.

Fig 1. HPLC chromatogram of B1W, Triphenyl-1-hexane and Ferulic acid standard solution.

Fig 2. The effects of B1W on (a) Micro-CT, (b) bone mineral density (BMD), bone volume fraction (BV/TV), and trabecular separation (Tb.Sp) of femurs.

Data are presented as the mean ±  SEM. ^##P <  0.01, ^###P <  0.001 versus the Sham group; *P <  0.05, **P <  0.01, ***P <  0.001, versus the OVX group.

Fig 3. The effect of B1W on (a) serum biomarkers, and (b) weight of uterus, body weight gain and food intake in OVX mice.

Data are presented as the mean ±  SEM. ^#P <  0.05, ^##P <  0.01, versus the Sham group; *P <  0.05, **P <  0.01, ***P <  0.001, versus the OVX group.

Fig 4. The effect of B1W on (a) osteoclast differentiation, (b) TRAP-positive multinucleated cells (3 or more nuclei), (c) viability of BMM, and (d) pits area.

Data from three independent experiments are presented as the mean ±  SEM values. Pit areas were quantified using the Image J program. *P <  0.05, ***P <  0.001, versus the 0 μg/mL B1W group.

Fig 5. The effects of B1W on RANKL-mediated mRNA expression of osteoporosis-related genes.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Data from three independent experiments are presented as the mean ±  SEM values. *P <  0.05, **P <  0.01, ***P <  0.001, versus the control group.

Fig 6. The effects of B1W on the expression of NFATc1 protein under RANKL.

The band was calculated based on the β-actin loading amount. Data from three independent experiments are presented as the mean ±  SEM values. **P <  0.01, ***P <  0.001, versus the control group.

Fig 7. The effects of B1W on MAP kinase signaling were analyzed by western blotting.

The phosphorylated and total forms of p38 and JNK were measured. All data were normalized by β-actin. Data from three independent experiments are presented as the mean ±  SEM. *P <  0.05, **P <  0.01, versus the control group.