Figure 1.
Acteoside inhibits RANKL-induced osteoclast differentiation from both BMMs and RAW264.7 cells.
BMMs were cultured for seven days in the presence of M-CSF (50 ng/ml) and RANKL (100 ng/ml) with increasing concentrations (0–20 µM) (A) or 5 µM acteoside (B). C and D, RAW264.7 cells were also exposed to the indicated acteoside concentrations in the presence of 100 ng/ml RANKL for seven days. After culturing, these cells were TRAP stained and the number of osteoclasts was counted. In the panels A and C, the results are expressed as a percentage of osteoclasts generated by M-CSF+RANKL (for BMMs) or RANKL alone (for RAW264.7 cells). Data are representative of three independent experiments (n = 4 per experiment). *p<0.05, **p<0.01, and ***p<0.001 vs. the cells cultured without acteoside.
Figure 2.
Acteoside attenuates RANKL-induced osteoclast differentiation without cytotoxic effects.
BMMs (A) or RAW264.7 cells (C) were cultured for 7 days in the presence of 50 ng/ml M-CSF, 100 ng/ml RANKL, or both with 10 µM of an anti-resorptive compound. TRAP staining was performed and the number of osteoclasts generated was calculated from 3 independent experiments (3 wells per condition were counted in each experiment). In addition, BMMs (B) and RAW264.7 cells (D) were incubated with 10 µM of each compound for 48 h and cell viability was measured by using WST-8 regent (n = 5 per each experiment). E, The concentration of the compounds to inhibit 50% of osteoclast formation was calculated from triplicate experiments. *p<0.05 vs. acteoside. ***p<0.001 vs. controls cultured with M-CSF and/or RANKL only. #p<0.05, ##p<0.01, and ###p<0.001 vs. cells cultured with acteoside. AC, acteoside; QC, quercetin; LU, luteolin; AP, apigenin; EG, EGCG.
Figure 3.
Acteoside prevents RANKL-induced pit formation in BMMs.
A, BMMs were pretreated with the indicated doses of acteoside for 2-coated 24-well plates and stimulated with 50 ng/ml M-CSF and 100 ng/ml RANKL for 7 days. Pit formation on the plate was observed under optic microscopy. B, BMMs were also cultured with M-CSF and RANKL in the presence of various acteoside concentrations (0–20 µM), and 7 days later, the resorbed area was quantified from 3 independent experiments and expressed as a percentage of the control (n = 4 per experiment). C, BMMs were treated with 10 µM acteoside 4 days after M-CSF and RANKL stimulation and incubated for addition-3 days followed by the analyses for TRAP staining and pit formation. The results in panel D show osteoclast and pit formation in BMMs 4 and 7 days after the osteoclastogenic induction without supplementation of acteoside. *p<0.05, **p<0.01, and ***p<0.001 vs. cells cultured with M-CSF and RANKL. #p<0.05 indicates significant difference between the experiments.
Figure 4.
Acteoside inhibits RANKL-induced MAPK activation in both BMMs and RAW264.7 cells.
BMMs (A) and RAW264.7 cells (B) were pretreated with the increasing doses (0–10 µM) of acteoside for 2 h followed by stimulation with 50 ng/ml M-CSF, 100 ng/ml RANKL, or both for 30 min. The phosphorylation of p38, ERK, and JNK was determined by immunoblot analysis using specific antibodies. The representative results from triplicate experiments are shown. C, Cells were stimulated with RANKL in the presence of acteoside and an immunometric assay was used to determine MAPK activities. The results were calculated from 3 independent experiments and are expressed as ng/ml (for p-p38 and p-ERK) or optical density (OD) at 450 nm (for p-JNK) normalized to control values (n = 4 per experiment). *p<0.05, **p<0.01, and ***p<0.001 vs. cells stimulated with RANKL.
Figure 5.
Acteoside suppresses NF-κB-DNA binding and phosphorylation of IκBα and the p65 subunit in RANKL-stimulated macrophages.
BMMs and RAW264.7 cells were pretreated with the indicated doses of acteoside for 2/ml RANKL for 30 min. A, NF-κB-DNA binding activity was determined and a representative result from triplicate experiments is shown. The phosphorylation of p65 and IκBα from BMMs (B) and RAW264.7 cells (C) was analyzed by immunoblotting. D, RAW264.7 cells were stimulated with 100 ng/ml RANKL for 24 h in the presence of 1 or 10 µM acteoside, and luciferase activity was measured. The result was calculated from 3 independent experiments and is expressed as a percentage of the control activity (n = 4 per experiment). ***p<0.001 vs. cells without RANKL or acteoside. #p<0.05 and ###p<0.001 vs. cells stimulated with RANKL alone. An NF-κB inhibitor peptide was used as a positive control.
Figure 6.
Acteoside attenuates inflammatory cytokine production and expression of c-Fos and NFATc1 in RANKL-stimulated macrophages.
A, BMMs were pretreated with increasing concentrations (0–10 µM) of acteoside for 2 h followed by stimulation with 50 ng/ml M-CSF and 100 ng/ml RANKL for 48 h. The levels of TNF-α, IL-1β, and IL-6 were determined using ELISA kits. BMMs (B) and RAW264.7 cells were also stimulated with 100 ng/ml RANKL in the presence of acteoside for 24 h and subjected to real-time RT-PCR analysis. In panels A, B, and C, the results were calculated from 3 independent experiments and are expressed as pg/ml or mRNA levels relative to the control (n = 4 per experiment). *p<0.05, **p<0.01, and ***p<0.001 vs. cells without RANKL and acteoside. #p<0.05, ##p<0.01, and ###p<0.001 vs. cells stimulated with RANKL. D, BMMs were pretreated with 10 µM acteoside for 2 h and stimulated with 100 ng/ml RANKL. At the indicated times (0–48 h after RANKL stimulation), c-Fos and NFATc1 protein levels were determined by Western blotting.
Figure 7.
Acteoside inhibits RANKL-mediated ROS production on osteoclast differentiation in BMMs.
The cells were incubated with 50/ml M-CSF for 24 h and then stimulated with 100 ng/ml RANKL for 1 h in the presence and absence of the indicated concentrations of acteoside. A, Cellular ROS were determined using flow cytometric analysis. B, DCF intensity was also calculated using WinMDI 2.9 program (n = 3). *p<0.05 and **p<0.01 vs. cells stimulated with M-CSF/RANKL.
Figure 8.
Oral administration attenuates the increases in serum biomarkers of bone turnover in ovariectomized animals.
Female ICR mice were ovariectomized (OVX) or given a sham operation (Sham). OVX mice were orally supplemented with 200 µl PBS containing 1 mM acteoside (AC mice) for 8 weeks and Sham and OVX mice were given the same volume of PBS for the same period. The serum levels of IL-6, IL-1β, ALP, calcium, TRAP, and OC were determined. Data are presented as mean ± SD (n = 8–10 mice/group). *p<0.05, **p<0.01, and ***p<0.001 vs. Sham group. #p<0.05 vs. OVX group.
Figure 9.
Acteoside restores fracture maximum force of the right mid-shaft of the femur and inhibits trabecular bone loss in ovariectomized animals.
At 1 day after the last acteoside treatment, the light femur of animals was collected and processed for various physiologic and morphometric analyses. A, Peak fracture force of mid-shaft of the femur was determined and expressed as ‘N’. B, Cortical bone of right femur was observed by optic microscopy. CB, cortical bone. C, Micro-CT images of the proximal femur. D, BMD (g/cm3), BV/TV (%), Tb.Th (µm), Tb.Sp (mm), and Tb.N (1/mm) were analyzed with micro-CT SkyScan CTAn software. Data are presented as mean ± SD (n = 8–10 mice/group). **p<0.01 and ***p<0.001 vs. Sham group. #p<0.05 vs. OVX group.
Figure 10.
Acteoside does not affect osteoblastogenesis of bone marrow cells.
A, Bone marrow cells were pretreated with 10 µM acteoside followed by treatment with DAG for 2 weeks and then stained with alizarin red. The dye absorbance (B) and calcium accumulation (C) were determined at the same time. D, After 2 days of DAG treatment, the mRNA levels of Runx2, osterix, BSP, and OC were measured by real-time RT-PCR. The results in panels B, C, and D was calculated from 3 independent experiments (n = 4 per experiment) and expressed as optical density or fold increase as compared to the controls without DAG or acteoside.