Isolation of bone marrow macrophages (BMMs) from mice
Ethical approval was approved by the Ethics Committee of Shengjing Hospital of China Medical University, Shenyang, China. ICR mice with ages of 4–6 weeks (Beijing Huakangkang Biotechnology Co., Ltd., Beijing, China) were sacrificed by skull relocation. Two hind leg bones were dissected out on a sterile station. Three ml of α-MEM complete media containing 10% serum was added to a 6 cm dish, and the cells in the bone marrow cavity were blew out into the dish three times using a 1 ml syringe. The cells were cultured in an incubator at 37 °C for 20 h (overnight). The cells in the super cell suspension was harvested, counted and used as primary BMMS.
Cell viability assay
BMMs (3 × 103) in 100 μl of DMEM containing 10% bovine serum were plated into each well of a 96-well plate and incubated in an incubator containing 5% CO2 at 37 °C overnight. Next day, polyphyllin VII (Solarbio, Beijing, China), which was dissolved with DMSO at stock solution and further diluted with medium to final concentration of 0 μM, 1 μM, 10 μM, 30 μM, or 50 μM, was added into the culture. The cells were further incubated for 9 days, and the cytotoxic effect of polyphyllin VII on the cells was tested using CCK-8 test kit (Dojindo, Japan) following the manufacture’s instruction.
Tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity test
BMMs were plated into a 96-well plate at a density of 1 × 104 cells/well and grouped as negative control group without any treatment, or experimental groups that were treated with 20 ng/ml of RANKL, 20 ng/ml of M-CSF and polyphyllin VII at the concentrations of 0, 1, 10, 30 and 50 μM. The media containing the corresponding induction reagents were changed once on the third day after seeding and once every other day during the following culture period. When osteoclasts were formed during 7–9 days in culture, the cells were fixed and stained with TRAP using commercial kit (Sigma-Aldrich, Cat. no. 387) following the manufacture’s instruction. The TRAP positive cells with multi-pseudopodia and multi-nuclei (> 3 nuclei) were counted. The activity of TRAP was determined using tartrate-resistant acid phosphatase assay kit (P0332, Beyotime Biotechnology, Shanghai, China) following the manufacture’s instruction.
Resorption pit formation assay
Resorption pit formation assay was performed on Corning 96-well plate (Corning, 3989, USA). Briefly, BMMs were seeded into a 96-well hydroxyapatite plate at a density of 1 × 103 cells/well, grouped as negative control group without any treatment, experimental groups that were treated with 20 ng/ml of RANKL, 20 ng/ml of M-CSF and polyphyllin VII at the concentrations of 0, 1, 10, 30 and 50 μM for 9 days. The media containing corresponding induction reagents were changed once on the third day after seeding and once every other day during the following culture period. When osteoclasts were induced (9 days induction), the media were discarded and replaced with 100 μL of 10% bleach. After standing at room temperature for 5 min, bleach was discarded, and cells were washed with ddH2O twice for 5 min each. They were air-dried at room temperature for 3–5 h. The resorption pits formed on the hydroxyapatite plate due to erosion by osteoclasts were observed and photographed under an invert microscope. Average areas of resorption lacunae per well was measured and expressed as pixel dimensions.
Assessment of intracellular ROS
BMMs were seeded into a 96-well plate at a density of 1 × 104 cells/well. The cells were grouped as negative control group without any treatment and experimental groups that were treated with 20 ng/ml of RANKL, 20 ng/ml of M-CSF and polyphyllin VII at the concentrations of 1, 10, and 30 μM. After 48 h induction, cells were washed with PBS twice, and 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was added in a final concentration of 50 μM. After incubation at dark at 37 °C for 30 min, cells were washed with PBS three times and treated with trypsin solution containing no phenol red. Cells were suspended in 500 μL of PBS, centrifuged at 1000 rpm, and re-suspended in 500 μL of PBS. The fluorescence from DCFH-DA bound to ROS was measured using flow cytometry.
Quantitative real-time RT-PCR (qRT-PCR)
Real time qRT-PCR was performed as previously reported procedure to quantify the mRNA of serum band 5 tartrate-resistant acid phosphatase (TRACP5), cathepsin K (CtsK), and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) . Briefly, total RNA was extracted using RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China). Reverse transcription was performed using Prime-Script RT reagent kit with gDNA eraser (Takara Biotechnology Co. Ltd., Dalian, China). Quantitative real time PCR was conducted using ABI 7500 (Applied Biosystem, Foster City, CA, USA) to determine mRNA expression of the target gene. Level of gene expression was expressed as relative to GAPDH and calculated using the 2-ΔΔCt method.
The primers used were as followings: Tracp5, forward primer: 5′-GTGCTGCTGGGCCTACAAAT-3′, reverse primer: 5′- TTCTGGCGATCTCTTTGGCAT-3′; Ctsk, forward primer: 5′- GAAGAAGACTCACCAGAAGCAG − 3′, reverse primer: 5′- TCCAGGTTATGGGCAGAGATT-3′; Nfatc1, forward primer: 5′-CCCGTCACATTCTGGTCCAT-3′, reverse primer: 5′-CAAGTAACCGTGTAGCTGCACAA-3′, and GAPDH, forward primer, 5′-ACCCAGAAGACTGTGGATGG-3′, reverse primer: 5′-TTCAGCTCAGGGATGACCTT-3′.
Active GTPase pull-down assay
Rac1-GTP pulled down assay was performed using Active GTPase Pull-down and Detection Kit (Thermos Scientific) following the manufacture’s instruction followed by immunoblotting for the target.
BMMs were pretreated with various concentrations of polyphyllin VII followed by inducing to osteoclasts with RANKL and M-SCF in the presence of varying concentrations of polyphyllin VII for 9 days. Cells were then harvested with RIPA containing inhibitors of proteases and phosphatase. Protein concentration was determined using BCA method. Proteins were differentiated by 10% SDS-PAGE gels, transferred onto PDF membrane, and immunoblotting was performed using the following primary antibodies: anti-PIK3, −p-PIK3 antibodies (Cell Signaling Technology); −TRAF6, −c-Src, −Rac1, and Nox1 antibodies (Abcam); and –ß-actin antibody (Proteintech). After reacting with appropriate 2nd antibodies, the protein bands were visualized by luminescent liquid and photographed. Band density was analyzed using Image J software.
Statistical analysis was performed using SPSS 17.0 software. One-way ANONA was used to analyze the differences among groups. If there was statistical difference between groups, the statistical significance was further determined by Tukey test, and comparison to control group was analyzed with Dunnett method. All data were expressed as mean ± SD. P < 0.05 was considered as statistically significant.