Isolation and culture of mouse BM-MSCs and osteoclasts.
For isolation of mouse BM-MSCs, 4 week-old C57BL/6 mice (Fourth Military Medical University, Xi’an, China) were sacrificed. Aseptically detached tibias and femurs, trimmed away excess soft tissues, and washed marrow cavity with PBS at 4 °C. Bone marrow cells were collected by centrifugation at 800 × g for 5 min and resuspended in 2 ml Dulbecco’s modified Eagle’s medium (DMEM) medium plus 10% fetal bovine serum (FBS) (v/v). The resuspended cells were seeded in a tissue culture flask for culture in a humidified 5% CO2 incubator at 37 °C. Then changed the medium every 2 days, and removed the floating cells. Upon reached 80–90% confluent, the adherent cells were passaged by trypsinization and subcultured. Cells after three passages were prepared for experiments. And bone marrow cells were obtained from the long bones of 6 week-old C57BL/6 mice (Fourth Military Medical University, Xi’an, China). Bone marrow cells cultured in the presence of M-CSF (50 ng/ml, PeproTech, Inc., Rocky Hill, NJ, USA) for 3 days to generate the bone marrow derived macrophages (BMMs). To examine osteoclast formation, BMMs were treated with Tanshinol at different concentrations in the presence of M-CSF (50 ng/ml) and RANKL (100 ng/ml, PeproTech, Inc., Rocky Hill, NJ, USA) in 96 well culture plates (Corning, MA, USA). Cells were fixed and stained for tartrate resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts. Experimental protocol involving animals was reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. All experiments were performed in accordance with the relevant guidelines and regulations of the Institutional Animal Care and Use Committee of the Fourth Military Medical University.
Induction of osteoblast differentiation and Alizarin red-sulfate staining
In the study, osteogenic differentiation of mouse BM-MSCs was induced by osteogenic medium (MUBMX-90021, Cyagen, USA). To examine the osteogenesis of mouse BM-MSCs, cells were seeded into a 96-well plate at ∼80 % confluent and the osteogenic medium added Tanshinol with different concentrations was changed every 2 days. After culturing 21 days, the cells were treated with Alizarin red-sulfate (ARS) staining. For Alizarin red-sulfate (ARS) staining, removed the medium, washed the cells with PBS for three times and fixed in 70 % methanol (v/v) at room temperature for 30 min. After washing three times with PBS, the cells were stained with 40 mM Alizarin red-sulfate (Sigma-Aldrich) aqueous solution, pH 4.2, for 20 min at room temperature using an orbital shaker at 100rpm. The cells were further rinsed with PBS to remove nonspecific staining and allowed to dry completely.
MTT method was used to analysis the impact on BMMs. BMMs were seeded in 96-well plates with the density of 1×105/ml, treated with Tanshinol at different concentrations. After 48 hours,10μL(5mg/ml) MTT per hole were added and incubated for 4 hours at 37℃.The cells were further added DMSO 150 μL per hole and dissolved for 10 minutes and then measured OD value at 490 nm absorbance.
Quantitative reverse transcription PCR (RT-PCR)
Quantitative RT-PCR experiments were conducted according to routine protocols. Total RNA was extracted with Trizol reagent (Invitrogen, USA), and cDNA was synthesized with a cDNA synthesis kit (Clontech, USA). Target genes were amplified from the synthesized cDNA and quantified by densitometry. The relative gene expression level was normalized against the β-actin transcript level. Each value represented the average of three independent experiments. The following primers were used for the amplification of target genes:
ALP forward primer 5′-AACCCAGACACAAGCATTCC-3′ and reverse primer 5′-GCCTTTGAGGTTTTTGGTCA-3′;
Collagen I forward primer 5′-ACAGCCGCTTCACCTACAGC-3′ and reverse primer 5′-TGCACTTTTGGTTTTTGGTCAT-3′;
β-catenin forward primer 5′-GGCAACCAAGAAAGCAAG-3′ and reverse primer 5′-CTGAACAAGAGTCCCAAGGAG-3′;
Axin2 forward primer 5′-TGACTCTCCTTCCAGATCCCA-3′ and reverse primer TGCCCACACTAGGCTGACA-3′;
C-fos forward primer 5′-CGGGTTTCAACGCCGACTA-3′ and reverse primer 5′-TTGGCACTAGAGACGGACAGA-3′;
CTSK forward primer 5′-GAAGAAGACTCACCAGAAGCAG-3′and reverse primer5′-TCCAGGTTATGGGCAGAGATT-3′;
TRAP forward primer 5′-CACTCCCACCCTGAGATTTGT-3′ and reverse primer 5′-CATCGTCTGCACGGTTCTG-3′;
NFATc1 forward primer 5′-TGGGAGATGGAAGCAAAGACTGA-3′ and reverse primer 5′-CATTGGCAGGAAGGTACGTGA A-3′;
β-actin forward primer 5′-CAATGTGGCCGAGGACTTTG-3′ and reverse primer 5′-CATTCTCCTTAGAGAGAAGTGG-3′.
Western blotting analysis
Proteins from osteoblasts and osteoclasts or the precursors with indicated treatments were extracted with ice-cold lysis buffer and centrifuged at 12,000 g for 10 min., and the resultant supernatant assayed using BCA protein assay kit standardized to BSA. Equal amounts of total protein (40μg) were loaded, separated by 12%SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in 5% TBST fat free milk for 2 hours, briefly washed, hybrid antibodies and then quantified using Imaging System Analysis software (VersaDoc Mp5000; Bio-Rad). Expression of β-actin protein served as a control.
Animals and Experimental Design
7 week-old C57BL/6 mice were purchased from Experimental Animal Research Center, the Fourth Military Medical University (Xi’an, China). The animals were maintained in individually ventilated cages (IVC) system (12 h light/dark cycle, 20.3–23.1 °C and 40–50% humidity) during the experiment cycle and fed with standard laboratory food and water ad libitum. The experimental animals were divided into five groups of six mice each.
Group I (Control group): sham operation group received normal saline (160 mg/ kg×day, i.g) for a period of 6 weeks;
Group II (Model group): OVX group received normal saline (160 mg/ kg×day, i.g) for a period of 6 weeks;
Group III (Tanshinol low-concentration group): mice treated with bilateral ovariectomy received Tanshinol (80 mg/ kgday, i.g) for a period of 6 weeks;
Group IV (Tanshinol medium-concentration group): mice treated with bilateral ovariectomy received Tanshinol (160 mg/ kg×day, i.g) for a period of 6 weeks;
Group V (Tanshinol high-concentration group): mice treated with bilateral ovariectomy received Tanshinol (240 mg/ kgday, i.g) for a period of 6 weeks.
At the end of the experimental period, mice were sacrificed by cervical decapitation. We used micro computed tomography (microCT) to examine the bone mineral density (BMD) and three-dimensional architecture parameters in trabecular bone of the distal femur.
All experiments were independently repeated for at least three times. All data were shown as mean ± SEM. ANOVA was used for statistical analysis, and P<0.05 was considered significant.