A total of 24 healthy male New Zealand white rabbits (age, 6 months; weight, 1–1.5 kg) were purchased from the Experimental Animal Center of Jilin City (Jilin, China). Rabbits were housed at 22–24°C (relative humidity, 55–70%) with natural light and air circulation, and were allowed free access to food and water. All animal procedures were conducted in strict accordance with the Animal Ethical Standard, and the present study was approved by the Experimental Animal Center of Beihua University, Jilin Province Ethics Committee (Jilin, China).

BMSCs were isolated from New Zealand white rabbits using the method described previously by Li et al (14). Initially, the femurs and tibias were removed, and flushed bone marrow cells were acquired via Percoll density gradient centrifugation (1.073 g/ml). Flushed bone marrow cells were washed with phosphate-buffered saline (PBS) and seeded into 25-cm² cell culture flasks. Flushed bone marrow cells were incubated with L-Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in an atmosphere containing 5% CO₂ for 48 h, and subsequently incubated with DMEM for 48 h. Cells were detached using 0.25% trypsin and 0.02% EDTA (Merck Millipore, Darmstadt, Germany) and centrifuged at 2,000 × g for 5 min. Suspended cells were gathered, seeded on 6-well plates at 1.5–2×10⁶ cells/well and incubated for two days.

BMSCs were fixed using 5% precooled paraformaldehyde for 30 min at 4°C and incubated with hematoxylin (Merck Millipore) for 10 min. BMSCs were washed with water for 10 min, and 95% ethyl alcohol and xylene were used to dehydrate and clear BMSCs, respectively. BMSCs were observed using light microscopy (D5300; Nikon Corp., Tokyo, Japan).

BMSCs (1–2×10⁶ cells or 1–2×10⁴ per well) were cultured in 6- or 96-well culture plates overnight at 37°C in an atmosphere containing 5% CO₂. Glycitin (Merck Millipore) was added to the wells at final concentrations of 0.01, 0.5, 1, 5 and 10 µM and cultured for 7 days.

In cells cultured in 6-well culture plates, BMSCs were determined using Oil Red O staining and observed via light microscopy at 510 nm. BMSCs were fixed using 5% precooled paraformaldehyde for 30 min at 4°C and stained with 0.6% (w/v) Oil Red O solution for 15 min at room temperature. Cells stained with Oil Red O were washed with water (3×5 min) to remove unbound dye, and culture dishes were stained with 1 ml isopropyl alcohol for 10 min.

In cells cultured in 96-well culture plates, BMSCs were determined via MTT assay. A total of 20 µl MTT (5 g/l) were added to each well and cultured for 4 h. The supernatant was removed and 200 µl dimethylsulfoxide were added to each well for 15 min. Optical density (OD) was measured using a microplate spectrophotometer (model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 570 nm. Proliferation rate was calculated using: OD treated / OD control × 100%.

Total RNA was extracted from BMSCs treated with glycitin (0, 0.5, 1 and 5 µM) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA (1–2 µg) was used to transcribe cDNA using a SYBR PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol PCR was performed on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR thermal cycling was performed as follows: Amplification at 94°C for 1 min, followed by 40 cycles of amplification at 94°C for 30 sec, annealing at 58°C for 45 sec, and extension at 72°C for 30 sec. Primers were designed as follows: Col I, forward 5′-TGACCTCAAGATGTGCCACT-3′ and reverse 5′-GGGAGTTTCCATGAAGCCAC-3′; and β-actin forward 5′-CGTGCGGGACATCAAGGA-3′ and reverse 5′-AGGAAGGAGGGCTGGAACA-3′. Subsequently, 7500 Fast Real-Time PCR system software was used to analyze crossing threshold (Cq) values using the second derivative maximum method (16).

BMSCs (1–2×10⁶ cells) were cultured in 6-well plates overnight at 37°C in an atmosphere containing 5% CO₂. Glycitin was added to the wells at final concentrations of 0, 0.5, 1 and 5 µM and cultured for 7 days. Cells were washed with ice-cold PBS and lysed via the repeated freeze-thaw method. Supernatant was analyzed using an ALP kit (Sangon Biotech Co., Ltd., Shanghai, China). ALP activity was calculated according to the formula: Treated group / control × 100%.

Proteins were extracted from BMSCs treated with glycitin (0, 0.5, 1 and 5 µM) by grinding with protease inhibitors. BMSCs were lysed using RIPA lysis buffer and protein content was measured using a micro-Bradford assay kit (Sangon Biotech Co., Ltd.). Protein samples (50–100 µg) were separated using 10–12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. Membranes were incubated with 5% non-fat milk for 2 h at room temperature, and were subsequently incubated with antibodies against TGF-β (1:500; sc-146), p-AKT (1:500; sc-135,650) and β-actin (1:1,000; sc-130,656; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C for 24 h. Membranes were washed three times with Tris-buffered saline with Tween-20 and subsequently incubated with an anti-rabbit secondary antibody (1:5,000; C2247; Applygen Technologies Inc., Beijing, China) at 37°C for 30 min at room temperature and were visualized with enhanced chemiluminescent reagent (ECL Plus; P0018A; Beyotime Institute of Biotechnology, Haimen, China). Proteins were quantified using Image Lab 4.1 (Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard deviation using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). Statistical differences were analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The constitutional formula of glycitin is displayed in Fig. 1. BMSCs appeared homogeneously mazarine blue, which demonstrated that BMSCs were successful extracted, indicating that BMSCs were successfully separated and cultivated (Fig. 2).

MTT assay was used to measure the effect of glycitin on the proliferation of BMSCs. As shown in Fig. 3, glycitin increased the proliferation of BMSCs, with statistical significance detected after treatment with 1 and 5 µM glycitin (P=0.0023 and P=0.0004, respectively).

As shown in Fig. 4, Oil Red O staining was observed in every group. A marked increase in red staining, and therefore osteoblasts, was detected following treatment with 1 and 5 µM glycitin.

In order to elucidate the effect of glycitin on the mRNA expression levels and activity of Col I and ALP, respectively, RT-PCR and ALP kits were used. The results demonstrated that administration of 1 and 5 µM glycitin significantly promoted Col I mRNA expression (P=0.0079 and P=0.0031, respectively) and ALP activity in BMSCs (P=0.0049 and P=0.0023, respectively; Fig. 5).

To confirm the effect of glycitin on TGF-β signaling in BMSCs, TGF-β protein expression of BMSCs was detected. The results indicated that pretreatment with 1 and 5 µM glycitin significantly enhanced TGF-β protein expression of BMSCs (P=0.0063 and P=0.0021, respectively; Fig. 6).

To investigate the effect of glycitin on p-AKT in BMSCs, p-AKT was measured using western blotting. The results demonstrated that 1 and 5 µM glycitin significantly increased the presence of p-AKT in BMSCs (P=0.0071 and P=0.0033, respectively; Fig. 7).