Cell culture
rBMSCs were purchased from Cyagen Biological Sciences (RASMX-01001; Santa Clara, CA, USA), and cultured in α-MEM (Hyclone, Pittsburg, PA, USA) containing 10% fetal bovine serum (Tbd, China) in a humidified chamber with 5% CO2 at 37°C. The medium was replaced every three days.
Cell-counting kit-8 (CCK8) assay
rBMSCs were seeded into 96-well plates at a density of 5×103 cells/well, then treated with different concentrations of ICA (10−4, 10−5, 10−6, 10−7, 10−8, 10−9 M). After culturing for 24 or 48 h, cell proliferation was measured using a CCK8 according to the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). Briefly, the culture medium was replaced with 100 µl α-MEM containing 10 µl CCK8, and the plates were incubated for 1 h at 37°C. Absorbance was measured at 450 nm using a multi-well spectrophotometer (BioTek, Synergy H4).
Alkaline phosphatase (ALP) activity
rBMSCs were seeded into 6-well plates at a density of 2×103 cells/well, and treated with different concentrations of ICA, as described in 2.2. Cells were treated with ICA for 3 and 7 days, and ALP activity assays were completed using ALP kits according to the manufacturer's instructions (Nanjing Jiancheng, Nanjing, China). Absorbance was measured at 520 nm using a multi-well spectrophotometer (BioTek, Synergy H4).
Alizarin red S staining
To determine calcium deposition, alizarin red staining was performed on day 7 of ICA treatment to evaluate the mineralized matrix. Cells were divided into control, 10−6 M ICA and β-estradiol (E2) treatment groups. After culturing for 7 days, the medium was removed, cells were washed three times with PBS and fixed in 95% ethanol for 10 min, then washed twice with PBS, and stained with 0.1% alizarin red at a pH of 7.2 (Sigma). After incubating for 5–10 min at room temperature, cells were again washed three times with PBS and examined via light microscopy.
Oil red O staining
rBMSCs were seeded into 6-well plates at a density of 2×103 cells/well, and divided into control, 10−6 M ICA and adipocyte-induced treatment groups. After rBMSCs were cultured for 14 days, oil red O staining was used to evaluate adipocyte differentiation of rBMSCs. Cells were rinsed twice with PBS, fixed with 4% paraformaldehyde for 15 min, then rinsed twice more with PBS. treated with 60% isopropanol for 1 min, and infusion with oil red O (Sigma-Aldrich) that was dissolved in 60% isopropanol for 20 min. This process was followed by three rinses with PBS. and then photographed.
Western blot analysis
rBMSCs were seeded into 10-cm culture dishes and divided into control, ICA, ICI and ICI+ICA treatment groups. Total protein was extracted with RIPA lysis buffer (P0013B, Beyotime, Haimen, China). Total protein was separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were washed in TBST, blocked with 5% skim milk and incubated overnight with anti-ERα (1:1,000, ab133467, Abcam, USA) and anti-ERβ (1:1,000, ab32063, Abcam), anti-RUNX2 (1:1,000, 12556S, Cell Signaling Technology, Danvers, MA, USA), anti-BMP-2 (1:1,000, ab14933, Abcam), anti-PPARγ (1:1,000, C26H12, Cell Signaling Technology), anti-CEBP/α (1:1,000, 2295, Cell Signaling Technology). Anti-GAPDH (1:1,000, D16H11, Cell Signaling Technology) was used as a loading control. The following day, membranes were incubated with anti-rabbit IgG secondary antibody (1:4,000, 7074P2, Cell Signaling Technology). Immunoreactive bands were visualized with an electrochemiluminescence reagent (5312-1; DOCLAB, Guangzhou, China) and quantified using Image J (NIH).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
rBMSCs were seeded into 60-mm culture dishes, and divided into four groups, as aforementioned. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was synthesized from mRNA using reverse transcriptase (AK4001; Takara, Shiga, Japan). After predenaturation at 95°C for 2 min, 40 RT-qPCR cycles were performed (95°C for 10 sec; and 60°C for 30 sec), followed by a final extension at 72°C for 10 min. Target gene expression was calculated using the formula 2−ΔΔCq (13). Gene expression was normalized to GAPDH. Gene primer sequences are shown in Table I.
Statistical analysis
Data were collected from three separate experiments and expressed as the mean ± standard deviation. The statistical differences were analyzed by one-way analysis of variance with Least Significant Difference and Student-Newman-Keuls post hoc tests using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.