BMSCs were isolated and cultured according to standard protocols (21). BMSCs were isolated from 80–100 g Sprague-Dawley rats. The cells were collected by flushing the rat femurs with phosphate-buffered saline (PBS) and culturing them in low-glucose Dulbecco's modified Eagle's medium (L-DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml; Shandong Lunan Pharmaceutical Factory) and streptomycin (100 U/ml; Shandong Lunan Pharmaceutical Factory) in a humidified atmosphere of 5% CO₂ at 37°C. The culture medium was replaced at 3-day intervals. The adherent cells were trypsinized with 0.25% trypsin-EDTA (Invitrogen; Thermo Fisher Scientific, Inc.) and subcultured in new flasks for further expansion when they reached 80–90% confluence. Cells in passage 3–5 were used for experiments. The experimental protocol was approved by the Institutional Animal Care Committee of Jiangsu University, Zhenjiang, China.

Adipogenesis: The BMSCs (2×10⁴) were seeded in 24-well plates in L-DMEM with 10% FBS. The medium was replaced with adipogenic induction medium (Cyagen Biosciences, Inc., Santa Clara, CA, USA) when cells attained 70–80% confluency. After cultivation for 21 days, adipogenic differentiation was assessed by the presence of intracellular lipid vesicles stained with Oil Red O (Cyagen Biosciences, Inc.) and visualized using an inverted microscope (TE300; Nikon Corporation, Tokyo, Japan).

Osteogenesis: BMSCs were plated in six-well plates at 1×10⁴ cells/cm² in L-DMEM with 10% FBS. After incubation overnight at 37°C with 5% CO₂, cells were cultured in osteogenic induction medium (Cyagen Biosciences, Inc.). After treatment for 14 days, osteogenic differentiation was detected by alkaline phosphatase staining following the manufacturer's instructions (Shanghai Sun Biotechnology Co., Ltd, Shanghai, China).

The phenotypes of BMSCs were analyzed with flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) after they were trypsinized with 0.25% trypsin-EDTA and stained with PE-conjugated monoclonal antibodies against CD29, CD90, CD105 or CD19 (FITC-conjugated) for 30 min at 4°C. FITC-IgG1 and PE-IgG1 (BD Biosciences) isotypic immunoglobulins were used as isotype controls.

The proliferative ability of BMSCs was determined with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at 3,000 cells per well and allowed to attach overnight at 37°C with 5% CO₂. Thereafter, cells were treated with different concentrations of IDB (0.5 to 60 µg/ml) for 0–96 h. MTT (20 µl) was added to each well for the last 4 h. The culture medium was then discarded and 150 µl of dimethyl sulfoxide (DMSO) were added to the plates to terminate the reaction. The plates were observed under a microscope after shaking for 10 min to solubilize the purple formazan crystals. The absorbance values were read at 490 nm using an enzyme-linked immunosor-bent plate assay reader (FLX800; BioTek Instruments, Inc., Winooski, VT, USA). The experiments were repeated three times for each experimental group.

BMSCs were plated in six-well plates at 1,000 cells per well and allowed to attach overnight at 37°C with 5% CO₂. The culture medium was then replaced with L-DMEM with 10% FBS containing 10, 20 or 30 µg/ml IDB for 14 days. The control group was treated with DMSO. The medium was changed at 3-day intervals. Finally, the cells were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 20 min. The cells were photographed and the number of colonies was counted for statistical analysis. The experiments were repeated three times for each experimental group.

The cells (2×10⁵) were seeded in 24-well plates in L-DMEM containing 10% FBS with 5% CO₂ at 37°C. After overnight incubation, cells were cultured with different concentrations of IDB (10, 20 and 30 µg/ml) for 24 h and morphological changes were observed using an inverted microscope under 40× magnification.

The cells were seeded in 24-well plates at a density of 20,000 cells per well. After culture with IDB at 10–30 µg/ml for 24 h, the cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 20 min and washed again with PBS. DAPI staining solution (final concentration 5 µg/ml) was added and cells were incubated in the dark at room temperature for 5 min. The DAPI staining solution was removed and the cells were washed three times with PBS, 5 min per wash. The stained cells were observed directly under a fluorescence microscope. Three non-overlapping fields were randomly captured per dish. The experiments were repeated three times for each experimental group.

BMSCs were plated in six-well plates at the third passage. After treatment with different concentrations of IDB (10, 20 or 30 µg/ml) for 24 h, the BMSCs were harvested, washed twice using cold PBS and stained with propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min at 4°C in the dark. We then used flow cytometry as described above to determine the cell cycle. The experiments were repeated three times for each experimental group.

Cells were seeded in six-well plates and incubated overnight. After treatment with IDB at 10, 20 or 30 µg/ml for 24 h, the cells were harvested, rinsed twice with cold PBS and incubated with PI and AnnexinV-FITC (BD Biosciences) for 20 min at room temperature. The stained cells were analyzed with flow cytometry as previously described. The experiments were performed in triplicate.

The migration of BMSCs was tested in transwell chemotaxis chambers (Corning Incorporated, Corning, NY, USA). Briefly, after culture with IDB (10, 20 or 30 µg/ml) for 24 h, 3×10⁴ cells were suspended in 200 µl serum-free medium and added to the upper chamber. Next, 600 µl of culture medium with 10% FBS was added to the lower chamber and the cells were incubated for 16 h. BMSCs which remained on the top side of the membrane were wiped off with cotton swabs. Cells which had migrated to the bottom side of the membrane were fixed in 4% paraformaldehyde and stained with crystal violet. We observed the stained cells under a microscope and selected at least six fields for each group. The experiments were repeated in triplicate for each group.

Expression of senescence-associated β-galactosidase (SA-β-gal) in BMSCs was detected using a SA-β-gal staining kit (Beyotime Institute of Biotechnology, Haimen, China). The cells were washed with PBS, fixed for 15 min using 4% formaldehyde and washed again with PBS. Subsequently, SA-β-gal stain solution was added and the plates were placed into a CO₂-free chamber. The percentage of blue-stained SA-β-gal-positive cells was counted in three random fields. Experiments were repeated at least three times.

BMSCs were cultured in IDB at 10–30 µg/ml for 24 h after cell density reached about 80%. The total protein from the BMSCs was obtained with RIPA lysis buffer containing protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA). The protein concentration of each sample was determined with a BCA protein assay kit (Invitrogen, Carlsbad, CA, USA). Proteins from each group were subjected to separation by SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated with monoclonal antibodies (mouse anti-rat) PCNA, cyclinD1, cyclinD3, Stat-3, Nanog, Oct-4, vimentin, E-cadherin, N-cadherin, Bcl-2, Bax, cleaved caspase-3 and monoclonal antibodies (rat anti-mouse) α-SMA at 4°C overnight. The membranes were washed with Tris-buffered saline/Tween three times and incubated with the appropriate secondary antibodies at 37°C for 1 h. The signals were visualized with Luminata Crescendo Western HRP substrate (Millipore, Billerica, MA, USA). All experiments were repeated at least three times.

Experiments were performed in triplicate and repeated at least three times independently. All data were expressed as means ± standard deviation (SD). Statistically significant differences between groups were assessed by analysis of variance using the GraphPad PrismV5.0 software program (GraphPad, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In primary culture, BMSCs displayed a polygonal spindly shape and contained one nucleus. After 7–10 days, they presented with a long spindle-shape and began to form colonies (Fig. 1A). The cells were induced to undergo adipogenic or osteogenic differentiation in the third generation, as shown by Oil Red O and alkaline phosphatase staining, respectively (Fig. 1B). According to flow cytometric analysis, BMSCs were positive for CD29, CD90 and CD105, but negative for CD19 (Fig. 1C).

The results of cell colony formation assays showed a dose-related increase in BMSC proliferation after incubation in increasing concentrations of IDB. Cell morphology changed from a broad flattened to a long spindle shape. At lower concentrations (10 µg/ml), IDB also had an obvious effect on the number and the morphology of colonies (Fig. 1D). Statistical analysis showed a significant increase in cell number between the control group and each treatment group with increasing IDB concentration (Fig. 1E).

MTT assays indicated that the viability of BMSCs in each treatment group was increased in a dose and time-dependent manner. At IDB concentrations of 10, 20 and 30 µg/ml, proliferative activity in the experimental groups was significantly higher than in the control group, but higher concentrations had no further effect on proliferation. Therefore, we selected the concentrations of 10, 20 and 30 µg/ml for the following experiments (Fig. 1F).

In the experimental groups, we observed that reticular formations of BMSCs appeared gradually and that the cells were tightly connected. The morphological shape of the cells became more like a slender spindle with increasing drug concentration. However, these changes were not found in the control group (Fig. 2A). The protective effects of IDB were further confirmed with DAPI staining. The cells were smaller and thinner in contrast to the DMSO group. In the IDB group, chromatin morphological changes were slightly improved (Fig. 2B). Using PI staining, the cell cycle in the presence of IDB was characterized by flow cytometric analysis. The results demonstrated that the cells in G1 phase in the treatment groups were marginally decreased while cells in S phase were slightly increased when compared with the control group. This suggested that IDB could promote the transformation of cells from G1 to S phase as shown by histogram analysis indicating statistically significant differences between the control and experimental groups (Fig. 2C, D). Considering the important role of IDB in cell apoptosis, we also used flow cytometry to detect apoptosis after treatment with IDB. The number of apoptotic cells in the IDB-treated groups was significantly less than in the DMSO group (Fig. 2E, F). Therefore, it appears that low concentrations of IDB may have an anti-apoptotic effect.

To determine whether the migratory ability of BMSCs was affected by IDB, we cultured the cells with increasing concentrations of IDB for 24 h. The results of cell migration assays indicated that IDB inhibited the migration of BMSCs when compared with the control group (Fig. 3A). Statistical analysis demonstrated a significant dose-related reduction in cell numbers after IDB exposure (Fig. 3B). The results of SA-β-gal staining showed that the experimental groups had fewer senescent cells and that senescence was decreased in a dose-dependent manner (Fig. 3C). Statistical analysis demonstrated that IDB significantly reduced the number of senescent cells (Fig. 3D).

To investigate the underlying mechanisms of IDB on BMSCs, we performed western blot experiments. We discovered that IDB reduced the expression of N-cadherin, vimentin and α-SMA but enhanced the expression of E-cadherin in vitro. These results are consistent with the role of these proteins in IDB-induced inhibition of migratory behavior in BMSCs (Fig. 4A-E). We also found that expression of Stat-3, Nanog and Oct-4 were increased after IDB exposure in a dose-dependent manner (Fig. 4F-I). Similarly, the expression of PCNA, cyclinD1 and cyclinD3 were increased after IDB treatment (Fig. 4J-M). Finally, we examined the important functions of Bcl-2, Bax and cleaved caspase-3 in the inhibition of cell apoptosis. Quantitative analysis revealed that IDB distinctly increased the ratio of Bcl-2 to β-actin while decreasing the ratio of Bax to β-actin (Fig. 4N-P). Moreover, the expression of cleaved caspase-3 protein, which is a key factor in the execution of apoptosis, was significantly suppressed as shown by statistical analysis (Fig. 4N-Q).