Contributed equally
Osteoporosis is a major public health problem, affecting over 200 million individuals worldwide (
As E2 is a well-documented factor for bone maintenance and hormone replacement therapy (HRT) has been demonstrated to possess beneficial effects on postmenopausal osteoporosis (
However, the anti-osteoporotic effects of
The roots of
For quantification of CW, 2,4-dihydroxyacetophenone was used as a standard. The content of 2,4-dihydroxyacetophenone was measured using a high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD; Agilent 1100 series; Agilent Technologies, Inc., Santa Clara, CA, USA). The extract was dissolved in 70% methanol and sonicated for 30 min. After filtering through a 0.45 µm filter membrane, an aliquot was injected in HPLC analysis. The column used was a Shiseido Capcell Pak C18 (250×4.6 mm, 5 µm; Shiseido Co., Ltd., Tokyo, Japan). The mobile phase consisted of 0.05% acetic acid in water and acetonitrile with 1.0 ml/min of flow rate at 30°C. As demonstrated in
A total of 36 female ICR mice aged 6 weeks (Raon Bio Animal, Inc., Yong-in, Korea) were provided free access to a standard chow diet (Orient Co. Ltd., Seongnam, Korea) and tap water. They were housed in a controlled environment (22±2°C, a relative humidity of 50±5% and a 12 h light:dark cycle). The animal studies were conducted in accordance with the rules and regulations established by the Institutional Animal Ethics Committee of the Kyung Hee University [KHUASP (SE) −15-079].
After acclimatization for 1 week, the mice, with the exception of the normal group, were surgically ovariectomized (OVX), then recovered and osteoporosis induced for 9 weeks. They were randomly divided into three groups (OVX + vehicle, OVX + E2, and OVX + CW). 17β-estradiol (E2; 10 µg/kg/day) was injected intraperitoneally to the OVX + E2 group as a positive control and 1 mg/kg/day CW orally administrated to OVX + CW group. Normal and OVX + vehicle mice were orally administrated vehicle (in PBS containing 1% DMSO). All mice were treated 5 times per week for 3 weeks, then sacrificed. The body weight was measured weekly. The blood sample was collected by cardiac puncture.
The epicondyles were removed and immediately fixed in 10% formalin for 18 h. Prior to dehydration, bone tissues were demineralized in 0.1 M ethylenediaminetetraacetic acid for 1 month. The sections of epicondyle were cut at a 5 µm thickness and stained with H&E or an Acid Phosphatase, Leukocyte TRAP kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Stained tissues were observed using Leica Application Suite microscope software (version 3.2.276.2; Leica Microsystems, Buffalo Grove, IL, USA).
Subsequent to sacrifice, the proximal femur was collected and cleaned without attached muscles and connective tissue. The sample was stored at −80°C in PBS until analysis. Dual-energy X-ray absorptiometry with a PIXImus instrument (Lunar Corp., Madison, WI, USA) was used for the determination of BMC and BMD.
The collected blood was centrifuged at 12,000 × g for 30 min at RT and the supernatant stored at −80°C until use. The concentration of serum osteocalcin was measured using Mouse Gla-osteocalcin high sensitive EIA kit (TaKaRa Bio, Inc., Otsu, Japan) according to the manufacturer's protocol.
Saos-2 cells (human osteosarcoma cell line; Korean Cell Line Bank, Seoul, Korea) were cultured with Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in 5% humidified CO2 atmosphere. Saos-2 cells were plated in 6-well culture plates at 0.8×105cells/well. CW (1, 10 and 100 µg/ml) in FBS-free DMEM medium was administered for 24 h.
RIPA buffer (50 mM Tris-HCl; pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) was used for uterus and Saos-2 cell protein extraction. The lysate (30 µg) was denatured with 2X loading buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then electrotransferred onto a PVDF membrane (Bio-Rad Laboratories, Inc.). Primary antibodies targeting osterix (cat. no. Ab94744; Abcam, Cambridge, UK), osteoprotegerin (cat. no. sc-11383; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) in TBS-T (1:1,000 dilution) were incubated overnight at 4°C and secondary antibody anti-mouse IgG (1:2,000 dilution; Cell Signaling Technology, Inc.) in TBS-T was incubated for 1 h at RT. The proteins were visualized using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Uppsala, Sweden). Visualized bands were quantified using a computerized densitometry system ImageJ (version 1.38e; National Institutes of Health, Bethesda, MD, USA). All samples were analyzed in triplicate.
Significance was determined by one-way analysis of variance and Dunnett's multiple comparison tests. P<0.05 was considered to indicate a statistically significant difference.
The BMC level in the OVX + vehicle group was significantly decreased to 0.034±0.003 g compared with the normal group (0.045±0.004 g). CW treatment significantly increased the level of BMC ~32% (0.045±0.004 g;
In the OVX + vehicle group, the pores within interstitial cells filling the lateral and medial epicondyles were markedly increased in comparison with the normal group. E2 injection as a positive control drug ameliorated histopathological changes of epicondyles. As recoveries in the E2 injected group, CW-treated mice demonstrated dense and well-formed bone marrow cells. Additionally, CW treatment reduced the bone marrow pores in the lateral and medial epicondyles (
The OVX + vehicle group demonstrated substantial increases of TRAP-stained multinucleated osteoclasts compared with normal mice. In lateral and medial epicondyles of the CW-administrated group, stained TRAP-positive cells were fewer compared with the OVX + E2 group (
The value of serum osteocalcin concentration was significantly lower in the OVX + vehicle group compared with the normal group. While the serum concentration of osteocalcin in normal group was 56.26±2.6 pg/ml, that in the OVX + vehicle group was 39.91±2.69 pg/ml; a significant ~29.06% decrease. Following treatment by CW, serum osteocalcin level demonstrated a 25.93% recovery (50.26±0.7 pg/ml;
When the cells were treated with various concentrations of CW, the expression of OPG was significantly increased (
Osteoporosis is a skeletal disorder characterized by low BMD level and microarchitectural deterioration of bone tissue (
Osteoporotic bone results from a homeostatic imbalance between bone resorption and bone formation (
Osteoclasts secrete TRAP, a biomarker of osteoclast differentiation, during bone resorption and its secretion is identified to correlate positively with resorptive behavior (
Osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein, is the most abundant non-collagenous extracellular matrix protein (
Taken together, treatment of CW maintained the bone integrity, inhibited the osteoclast formation and induced the osteoblast differentiation. These results suggest that CW has ameliorative effects on osteoporosis and could be used as a treatment for osteoporosis. Further studies are required to clarify the molecular mechanisms through which CW ameliorates osteoporosis.
This work was supported by Samik Dairy & Food Co., Ltd. (Seoul, Korea).
Standardization of CW using high-performance liquid chromatography. Chromatograms of standard 2,4-dihydroxyacetophenone (upper panel) and CW (lower panel). CW,
The effects of CW on bone mineral content and bone mineral density of femurs. Results are presented as the mean ± standard error. #P<0.05 and ###P<0.001, normal group vs. OVX + vehicle group. *P<0.05, **P<0.01 and ***P<0.001, OVX + CW or E2 group vs. OVX + vehicle group. CW,
The effect of CW on pores within (A) interstitial cells filling the lateral and medial epicondyles and (B) osteoclast population. Sections in panel A were stained with hematoxylin and eosin (magnification, ×200), and in panel B with tartrate resistant acid phosphatase (magnification, ×400). Red arrows indicate multi-nuclei osteoclasts. CW,
The effects of CW on serum osteocalcin concentrations. Results are presented as the mean ± standard error. ###P<0.001, normal group vs. OVX + vehicle group. **P<0.01, OVX + CW or E2 group vs. OVX + vehicle group. CW,
The effects of CW on the expression levels of OPG and osterix in Saos-2 cells. Results are presented as the mean ± standard error. ***P<0.001, non-treated vs. CW-treated cells. CW,