Male 4-week-old Sprague-Dawley rats (n=10) weighing 80–100 g were used in the present study and were obtained from Guangzhou Laboratory Animal Center, Guangzhou University of Chinese Medicine (Guangzhou, China). Low glucose Dulbecco's modified Eagle's medium (DMEM) and PBS were acquired from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). H₂O₂ was purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Basal medium of Sprague-Dawley rat MSCs, fetal bovine serum (FBS), glutamine, penicillin-streptomycin and trypsin were purchased from Cyagen Biosciences, Inc. (Guangzhou, China). MTT and dimethyl sulfoxide were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Gigantol was purchased from the National Institute for Food and Drug Control (cat. no. 111875; Beijing, China). The chemical structure of gigantol is presented in Fig. 1A. Annexin V-fluorescein isothiocyanate (FITC) apoptosis, Hoechst 33258 and reactive oxygen species (ROS) assay kits were provided by Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). The PI3K/Akt inhibitor LY294002 was purchased from Selleck Chemicals (Houston, TX, USA). All other chemicals were of analytical grade.

MSCs were immediately isolated from the Sprague-Dawley rats as previously described, with minor modifications (18). Briefly, Sprague-Dawley rats were sacrificed by cervical dislocation. The experimental procedures were approved by the Laboratory Animal Committee of Guangdong Province (Guangzhou, China). All treatments on animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals (19). The femurs and tibias of rats were carefully cleaned of adherent soft tissue, the marrow was harvested and flushed with serum-free DMEM with 1% penicillin-streptomycin until the bone washed pale. Cells were resuspended in DMEM medium with 10% FBS and 1% penicillin -streptomycin of Sprague-Dawley rBMSCs at 37°C with 5% CO₂ After being allowed to attach for 24 h, hematopoietic and non-adherent cells were removed by changing the medium. Subsequently, rBMSCs were harvested for the experiments described below between the second and third passage. Cells were pretreated with gigantol for 12 h followed by treatment with H₂O₂ for 2 h, both at room temperature. To determine the effect of LY294002, cells were pretreated with LY294002 (25 µmol/l) for 1 h at room temperature, followed by the treatments with gigantol and H₂O₂.

Cells were seeded in 96-well plates (1×10⁵ cells/ml) for 24 h at room temperature. To determine the effects of gigantol and H₂O₂ on rBMSC viability, cells were treated with 1, 10, 40, 80 and 100 µM gigantol for 12 h, or 400, 500, 600, 700, 800 and 900 µM H₂O₂ for 2 h, respectively. As a control, cells were treated with DMEM medium only. Furthermore, in another cell viability assay, cells were pretreated with different concentrations of gigantol (1, 10, 40, 80 and 100 µM) for 12 h followed by treatment with 600 µM H₂O₂ for 2 h, both at room temperature. Subsequently, 20 µl MTT was added to each well and incubated at 37°C for 4 h prior to removal and addition of 100 µl dimethyl sulfoxide. The absorbance value was measured in a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm. Statistical analysis was performed on absorbance value readings.

Cells were cultured in 24-well plates (5×10⁵ cells/well) and treated with 80 µM gigantol for 12 h followed by the addition of 600 µM H₂O₂ for 2 h. Cells in the H₂O₂ group were treated with 600 µM H₂O₂ only. Cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS twice prior to staining with Hoechst 33258 for 5 min at 4°C in the dark. Condensed nuclei and cell shrinkage were observed using an inverted and fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). A bright blue stain indicated apoptotic cell nuclei.

Cells were cultured in 6-well plates (1×10⁶ cells/well) and treated with 80 µM gigantol for 12 h followed by the addition of 600 µM H₂O₂ for 2 h, both at room temperature. Cells in the H₂O₂ group were treated with 600 µM H₂O₂ only. Cells were stained with 10 µM 2′7′-dichlorofluorescin diacetate (DCFH-DA) diluted with serum-free medium at 37°C for 20 min and later washed with serum-free medium three times. Fluorescence intensity was analyzed using a microplate reader (Bio-Rad Laboratories, Inc.) at excitation and emission wavelengths of 488 and 525 nm, respectively. Images were captured using a fluorescence microscope (Leica Microsystems GmbH). The absorbance values were obtained for statistical analysis.

Cells were seeded in 6-well plates (1×10⁶ cells/well) for 24 h and and treated with 80 µM gigantol for 12 h followed by the addition of 600 µM H₂O₂ for 2 h. Cells in the H₂O₂ group were treated with 600 µM H₂O₂ only. Subsequently, cells were harvested and washed twice using PBS, and were resuspended in 500 µl binding buffer. Annexin V-FITC stock (5 µl) and propidium iodide solution (5 µl) was added to the cells and incubated for 10 min at room temperature in the dark, and immediately analyzed using flow cytometer (BD FACSCanto II). The percentage of apoptotic cells was obtained for statistical analysis.

Cells were seeded in 6-well plates (1×10⁶ cells/well) for 24 h and treated with 80 µM gigantol for 12 h followed by the addition of 600 µM H₂O₂ for 2 h, both at room temperature. Cells in the H₂O₂ group were treated with 600 µM H₂O₂ only. Cells in the gigantol + H₂O₂ + LY294002 group were pretreated with LY294002 (25 µmol/l) for 1 h prior to gigantol with H₂O₂ treatment. Subsequently, cells were washed with PBS and lysed in cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with protein inhibitor. Cellular proteins were collected and their concentrations were determined using a Bradford assay. Equal amounts of protein (40 µg/lane) were separated on 15% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes via electrophoresis. After blocking with tris-buffered saline (TBS) containing 5% skimmed milk and 0.05% Tween-20 for 1 h at room temperature, the membranes were incubated with the following primary antibodies: p-Akt (ser 473; cat. no. Sc7985r; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Akt (ser 473; cat. no. Sc8312; 1:200; Santa Cruz Biotechnology, Inc.), B-cell lymphoma-2 (Bcl-2)-associated X (Bax; cat. no. 2772; 1:1,000; CST Biological Reagents Co., Ltd., Shanghai, China), Bcl-2 (cat. no. 2872; 1:1,000; CST Biological Reagents Co., Ltd.), Caspase-3 (cat. no. 9662; 1:1,000; CST Biological Reagents Co., Ltd.), Caspase-9 (cat. no. 9504; 1;1,000; CST Biological Reagents Co., Ltd.) and β-actin (cat. no. sc58673; Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing with TBS three times, the membranes were incubated with goat anti-rabbit immunoglobulin G antibodies conjugated with horseradish peroxidase (cat. no. 111-035-003; 1:1,000; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature. Following three washes with TBS-Tween-20, the intensity of bands was visualized using an enhanced chemiluminescence western blotting kit (Merck KGaA) and quantified by densitometric analysis with ImageJ software (version 3.0; National Institutes of Health, Bethesda, MD, USA).

All experiments were conducted at least three times. Data are presented as the mean + standard error of the mean. Differences among groups were analyzed by one-way analysis of variance, followed by Dunnett's post-hoc test, using SPSS version 20 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine an appropriate concentration of gigantol, cells were treated with gigantol (1, 10, 40, 80 and 100 µM), and the results indicated that none of these concentrations exhibited a damaging effect on cell viability (Fig. 1B). Cell viability was reduced in a dose-dependent manner when treated with 400, 500, 600, 700 and 800 µM H₂O₂ for 2 h, compared with the control group. H₂O₂ at the concentration of 600 µM significantly reduced cell viability compared with the control by 51.6±3.2% (Fig. 1C). In addition, results in Fig. 1D demonstrated that gigantol significantly increased the cell viability of rBMSCs in a dose-dependent manner compared with cells treated with H₂O₂ only. Furthermore, pretreatment with 80 µM gigantol significantly enhanced cell viability compared with the H₂O₂ only group (Fig. 1D). Concentrations of gigantol >80 µM reduced the stimulatory effect. Therefore, 600 µM H₂O₂ and 80 µM gigantol were selected for the following experiments.

Following treatment with H₂O₂, apoptosis-associated morphology was observed in rBMSCs, including detachment, irregular shape and nuclear shrinkage. However, the number of apoptosis-like cells decreased in the group pretreated with gigantol, which indicated a potential protective effect of gigantol from apoptosis induction (Fig. 2A and B).

Cellular oxidative stress was examined by a DCFH-DA assay. The results demonstrated that, in the H₂O₂-treated group, a significant increase in 2′,7′-dichlorofluorescein fluorescence was observed (Fig. 2C and D). However, pretreatment with gigantol significantly reduced the intracellular production of ROS compared with the H₂O₂-treated group (Fig. 2C and D).

Cell apoptosis was analyzed using an Annexin V and propidium iodide double-staining assay by flow cytometry. The percentage of apoptotic cells in Q2 and Q4 increased from 0.5±0.45% in the control group to 49.5±3.30% in the H₂O₂ group, while apoptosis was significantly reduced to 23.4±2.06% in the gigantol + H₂O₂ group, compared with the H₂O₂ only group (Fig. 3).

The results of western blot analysis demonstrated that H₂O₂ treatment reduced the protein levels of phosphorylated (p)-Akt and the antiapoptotic protein Bcl-2 (Fig. 4A and B), and increased the levels of the proapoptotic proteins Bax, caspase-3 and caspase-9 (Fig. 4B-D). However, gigantol pretreatment lowered the caspase-3, caspase-9 and Bax levels, and increased the levels of p-Akt and Bcl-2, compared with the H₂O₂ only group (Fig. 4). Furthermore, LY294002 (a PI3K inhibitor) significantly inhibited the protective effect of gigantol against H₂O₂-induced apoptosis by increasing the levels of caspase-3, caspase-9 and the ratio of Bax/Bcl-2, and decreasing the ratio of p-Akt/Akt (Fig. 4).