A total of 6 male C57BL/6 mice (8 weeks old) were obtained from the Experimental Animal Center of Xinxiang Medical University (Xinxiang, China). The study protocol and animal use were approved by the Ethics Committee of Xinxiang Medical University. Animal treatment was strictly performed according to the Guidelines of the Ministry of Science and Technology of the People's Republic of China (approval no. 2006-398).

BMSCs were isolated and cultured according to a recently published protocol (21). The third-passage BMSCs were exposed to 0, 10 and 20 ng/ml recombinant mouse TGF-β1 (Cell Signaling Technology, Inc., Danvers, MA, USA) for 24 h, and cell proliferation, apoptosis and mitochondrial ROS were subsequently measured.

BMSC viability was measured with an MTT assay kit (American Type Culture Collection, Manassas, VA, USA) following exposure to different concentrations of TGF-β1. The third-passage BMSCs (3×10³ cells/well) were placed in 96-well plates and cultured with Dulbecco's modified Eagle's medium (DMEM) without serum overnight. The next day, the medium was replaced with complete DMEM (with 15% serum) supplemented with 0, 10 and 20 ng/ml TGF-β1, and the cells were cultured for an additional 24 h. A total of 10 µl MTT reagent was then added to each well and the plates were incubated at 37°C for 2 h. Following incubation, MTT detergent reagent (100 ml) was added to each well and the plates were incubated at room temperature for 2 h in the dark. Finally, absorbance at 570 nm was read using a plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell apoptosis was measured using DAPI (Beyotime Institute of Biotechnology, Shanghai, China) staining. BMSCs (1×10⁵ cells/well) were seeded in 24-well plates with round coverslips and exposed to 0, 10 and 20 ng/ml TGF-β1 for 24 h. Following washing with phosphate-buffered saline (PBS), the cells were fixed with buffered paraformaldehyde and incubated with 0.1% Triton™ X-100 (Bao Biological Engineering Co., Ltd., Dalian, China) for 10 min at room temperature. The cells were treated with DNase-Free RNase (50 mg/ml) for 3 h at 37°C and then stained with 5 µM DAPI for 5 min at room temperature. Cell apoptosis was assessed under a fluorescence microscope (Olympus Corp., Tokyo, Japan). The cells with a condensed nucleus or a nucleus with irregular edges were considered to be apoptotic.

Following treatment with different concentrations of TGF-β1 for 24 h, proteins were extracted from the BMSCs using a protein extraction kit containing 20 ml 5X lysis buffer, 0.5 ml protease inhibitor, 0.5 ml phosphatase inhibitor and 0.5 ml phenylmethylsulfonyl fluoride (Bao Biological Engineering Co., Ltd.). Protein concentrations were measured using a standard Bradford protein assay method‥ Western blot analysis was performed according to a recently published protocol (22). In brief, 20 µg proteins were mixed with equal volume of 2X loading buffer (Bao Biological Engineering Co., Ltd.) and heated at 95°C for 5 min. Proteins were separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bao Biological Engineering Co., Ltd), then transferred onto polyvinylidene fluoride membranes (Bao Biological Engineering Co., Ltd). The membranes were blocked with 5% non-fat milk (Bao Biological Engineering Co., Ltd) in Tris-buffered saline with Tween-20 (TBS-T; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and incubated with the following primary antibodies: Rabbit polyclonal IgG anti-B-cell lymphoma 2 (Bcl-2; 1:1,000; sc-492; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal IgG anti-Bcl-2-associated X protein (Bax; 1:1,000; sc-493; Santa Cruz Biotechnology, Inc.), rabbit polyclonal IgG anti-Annexin V (1:2,000; ab14196; Abcam, Cambridge, MA, USA) or mouse monoclonal IgG anti-β actin (1:2,000; sc-47778; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following washing with TBS-T three times, the membranes were incubated with donkey anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (1:10,000; sc-2313; Santa Cruz Biotechnology, Inc.) or donkey anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody (1:10,000; sc-2314; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The immunoreactive bands were visualized with an Enhanced Chemiluminescence Western Blotting Substrate Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The mean expression levels of Annexin V, Bax and Bcl-2 in each group were calculated using Image J software [National Institutes of Health (NIH), Bethesda, MD, USA].

Mitochondrial ROS were measured by flow cytometry following staining with MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen Life Technologies). The BMSCs were plated in 24-well plates, treated with different concentrations of TGF-β1 for 24 h and then detached using 2.5% trypsin digestion. Following digestion, the cells were suspended in warm PBS and incubated with 5 µM MitoSOX reagent for 10 min at 37°C in the dark. The cells were then washed thrice with PBS, and the mitochondrial ROS levels were analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA).

Statistical analysis was performed with SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Univariate comparisons of means were performed using one-way analysis of variance followed by Tukey tests. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control, treatment with 10 and 20 ng/ml TGF-β1 for 24 h significantly decreased BMSC viability (Fig. 1).

An increased number of cells with a condensed nucleus or a nucleus with irregular edges (DAPI staining) was observed in the TGF-β1 (10 and 20 ng/ml)-treated cells compared with the control cells, which shows that TGF-β1 can stimulate BMSC apoptosis (Fig. 2). The mean rate of apoptosis in each group was calculated, and the results are shown in Table I.

Annexin V is an anticoagulant protein that is typically used as an indicator of cell apoptosis. Bcl-2 is an important member of the Bcl-2 family of regulator proteins that inhibits cell apoptosis (23). Bax is an antagonist of Bcl-2 that promotes cell apoptosis (23). Western blotting showed that treatment with 10 and 20 ng/ml TGF-β1 significantly increased the expression of Annexin V and Bax but decreased the expression of Bcl-2 (Fig. 3). The average expression levels of Annexin V, Bax and Bcl-2 in each group were calculated using Image J software (NIH) and are listed in Table II.

Mitochondrial ROS are strong inducers of cell apoptosis (24). It has been indicated that TGF-β1 can induce mitochondrial ROS production in cell lineages other than BMSCs (19,25). Flow cytometric data showed that treatment with TGF-β1 (10 and 20 ng/ml) also markedly increased mitochondrial ROS levels in BMSCs (Fig. 4).