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Introduction

Bone marrow stem cells (BMSCs) are considered as the best 'seeds' in cell replacement therapy (CRT) and tissue engineering due to their strong differentiation potential, easy availability and amplification, and absence of rejection in autotransfusion. BMSCs have wide applications in bone and cartilage reconstruction (1,2) and the repair and therapy of bone marrow injury (3), hypoxic-ischemic nerve cells (4) and myocardial cells (5,6). Previous studies have demonstrated that the growth and differentiation potential may be affected either due to the accumulation of reactive oxygen species (ROS) in the region of BMSCs transplantation caused by ischemic injury (7) or due to the increased level of ROS resulting from natural aging or estrogen deficiency (8,9). Therefore, the effect of BMSCs transplantation in CRT and tissue engineering is greatly impaired.

Elevation of ROS levels is the major reason for mitochondrial swelling, decline of mitochondrial membrane potential, calcium overload and the release of precursor proteins in mitochondrial death pathway (10,11). Oxidative stress leads to apoptotic injury in BMSCs. Therefore, the improvement of the survival of BMSCs in the transplantation region by anti-oxidative and anti-apoptotic therapies is key to the success of CRT and tissue engineering.

Previous studies indicated that estrogen antagonizes the apoptosis of BMSCs under oxidative stress by protecting the mitochondrial membrane integrity (12–14). Phytoestrogen possesses estrogen-like activity and exhibits the function of clearing free radicals and anti-oxidative effects in cellular experiments (15,16). Ginsenoside Rg1 is a representative monomer in panaxatriol saponins and the primary active compound in ginseng. As a type of phytoestrogen, ginsenoside Rg1 demonstrates anti-aging, anti-oxidative and anti-apoptotic abilities in nerve and cardiovascular cells (17–20). The present study hypothesized that ginsenoside Rg1 may antagonize the apoptosis of BMSCs under oxidative stress by protecting mitochondrial membrane integrity.

The phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) pathway is one of the primary signal transduction pathways inhibiting cell apoptosis and promoting cell survival (21). The PI3K/Akt pathway has an anti-apoptotic effect via the phosphorylation of downstream protein Bad and the activation of B-cell lymphoma 2 (Bcl-2) (22). Previous studies demonstrated that activation of the PI3K/Akt pathway regulates the H2O2-induced apoptosis of rat BMSCs (rBMSCs) (23–27). The current study hypothesized that the antagonistic effect of ginsenoside Rg1 against the apoptosis of BMSCs induced by oxidative stress may be associated with the activation of the PI3K/Akt pathway.

A model of H2O2-induced oxidative injury in rBMSCs was developed in order to verify the above hypothesis. The protective effect of ginsenoside Rg1 against H2O2-induced oxidative injury in rBMSCs was observed. In addition, the possible associations with the PI3K/Akt pathway were investigated to understand the application of BMSCs in CRT and tissue engineering.

Materials and methods

Materials

Healthy female Sprague-Dawley rats (n=10, weight, 180±20 g; age, 4 weeks) were provided by Nanjing Medical University (Jiangsu, China). Ginsenoside Rg1 was purchased from Shanghai Oriental Pharmaceutical Co., Ltd. (Shanghai, China). Gibco low-glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Cell Counting Kit-8 (CCK-8) assay kit was supplied by Dojindo Laboratories (Kumamoto, Japan) and polyvinylidene fluoride membrane by Roche Diagnostics (Basel, Switzerland). Bax, Bcl-2, phosphorylated (p)-Akt and Akt primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). This study was approved by the ethics committee of Nanjing Medical University.

Isolation, culture and identification of rBMSCs

The rats were sacrificed by cervical dislocation and soaked in 70% alcohol for 20 min. The tibia and femur were harvested under aseptic conditions, and the diaphysis was severed using sterilized ophthalmic scissors. The marrow cavity was flushed repeatedly with phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA) solution, and then transferred to a centrifuge tube for centrifugation for 5 min at 120 × g for 5 min. The supernatant was discarded, and the precipitate was resuspended in 85% low-glucose DMEM [15% FBS + penicillin/streptomycin (Hangzhou Sijiqing Biological Engineering Materials, Co., Ltd., Hangzhou, China)]. The cells were then seeded at 1×106 cells/cm2 and cultured in a 37°C, 5% CO2 incubator. Half a volume of the medium was replaced 24 h later, and the full volume was replaced 2–3 days later. When the cells reached 80% confluency, they were passaged by digestion using 0.25% trypsin. The cells of the third generation were collected to detect purity and were free from the non-adherence spherocytes, thus the purified rBMSCs were obtained.

Determination of final concentrations of H2O2 and ginsenoside Rg1 using the CCK-8 assay

rBMSCs of the third generation were prepared into 1×105 cells/ml single-cell suspension and seeded to 96-well plates (104 cells/well). Following cell adherence to the wall of the plate, the cells were starved for 24 h by adding 100 µl serum-free culture medium. Five wells were randomly selected, and culture media containing different concentrations of H2O2 (0, 200, 400, 600 and 800 µM; Beijing Haiderun (Sea Derun) Pharmaceutical Co., Ltd., Beijing, China) was added to treat the cells for 6 h. Cells were then incubated at 37°C for 30 min with 10 µl CCK-8 solution. The absorbance was measured at 450 nm (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

To determine the final concentration of H2O2, cells were starved for 24 h using the method described above and 4 wells were randomly selected. Ginsenoside Rg1 of different concentrations (0, 0.1, 1 and 10 µM) was added into each well to treat the cells for 24 h, followed by 800 µM H2O2 treatment for 6 h. One group was randomly selected as the control group, for which no treatment was performed. The absorbance was measured using the CCK-8 solution as described above.

Grouping

Cells of third generation reaching the logarithmic growth phase were divided into 4 groups as follows: Control, untreated; H2O2-treated, addition of 800 µM H2O2 to induce oxidative injury; ginsenoside Rg1-treated, 10 µM ginsenoside Rg1 for 24 h, followed by 800 µM H2O2 for 6h; and Akt pathway blockage group, blockage achieved by addition of 25 µM LY294002 (Cell Signaling Technologies, Inc., Boston, MA, USA) for 1 h, followed by ginsenoside Rg1 and H2O2 treatments.

Detection of cellular apoptotic rate by TUNEL staining

The rBMSCs of the third generation were seeded into a 24-well plate. The cells in sub-aggregation state were starved for 24 h using the serum-free culture medium. When the cells in each treatment were dried, they were fixed in 4% paraformaldehyde (Wuhan GoodBio Technology, Co., Wuhan, China) for 1 h, and sealed for 10 min using the confining liquid (3% H2O2, dissolved in methanol). Subsequent to transparentization for 2 min using 0.1% Triton X-100 (Biosharp, Hefei, China), the cells were sealed for 1 h in the TUNEL reaction mixture at 37°C in a dark box. The cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology, Haimen, China) for 5 min, and the fluorescence was detected Nikon eclipse Ti (Tokyo, Japan). TUNEL (Roche, Indianapolis, IN, USA) staining is an intuitive method to detect apoptosis. With DAPI-TUNEL double staining, the cells are counted directly, which enables the comparison of cellular apoptotic rate between the samples. The blue fluorescence indicates that the cells were stained by DAPI and the green fluorescence indicates that the cells were stained by TUNEL.

Detection of cellular apoptosis using flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) double staining

rBMSCs of the third generation were inoculated into a 6-well plate. Following cell adherence to the walls of the plate, cells were divided into the 4 treatment groups. Following treatment, cells were collected and centrifuged for 5 min at 270 × g. The supernatant was discarded and cells were washed twice with PBS. Cells were then resuspended in 500 µl of binding buffer, and 5 µl FITC-labeled Annexin V (20 µg/ml) and 5 µl PI (50 µg/ml) (all from; BD PharMingen,San Diego, CA, USA) were added to the solution. The reaction was conducted in the dark for 15 min, and cell apoptosis was detected by BD FACS ARIA II flow cytometry (BD Biosciences, San Jose, CA, USA).

Detection of p-Akt, Bcl-2, Bax and cleaved caspase-3 protein expression levels by western blotting

Western blotting was conducted to detect apoptosis-associated proteins and p-Akt levels in H2O2-treated rBMSCs with Rg1 pretreatment. Subsequent to each treatment, cells were washed twice with PBS, and the pre-cooled cell lysis buffer was added. The reaction proceeded on ice for 5 min and the cells were then scraped off. The products of lysis were centrifuged for 15 min at 4°C at 14,000 × g. The supernatant was collected to determine the protein concentration using the Bicinchoninic Acid assay kit (Beyotime Institute of Biotechnology). Equal quantities of protein sample were subjected to 10% sodium dodecyl sulfate-polyacrylimide gel electrophoresis 80 mA for first 30 min then 120 mA for last 90 min (Beyotime Institute of Biotechnology). Following electrophoresis, proteins were transferred to a nitrocellulose membrane (Biosharp), which was sealed with 5% nonfat milk powder containing 0.05% Tween-20 with Tris-buffered saline (TBST; Biosharp) at room temperature for 1 h. The membranes were then incubated with the following antibodies: Rabbit anti-p-Akt (1:1,000; cat. no. #9271), polyclonal anti-Akt (1:1,000; cat. no. #9272), polyclonal anti-Bcl-2 (1:1,000; cat. no. #2876), polyclonal anti-Bax (1:1,000; cat. no. #2772) and monoclonal anti-caspase-3, (1:1,000; cat. no. #9665) (all from Cell Signaling Technologies, Inc.) primary antibodies at 4°C overnight. Membranes were washed 3 times with TBST for 15 min, incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibodies (1:2,000; cat. no. #7074; Cell Signaling Technologies, Inc.) for 2 h, followed by 3 washes with TBST for 10 min. Enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) was utilized to visualize the proteins. Images were captured using an imaging system (UVP Inc., Upland, CA, USA). The images were analyzed using Image Lab software v2.0.1 (Bio-Rad Laboratories, Inc.). The ratio of absorbance of the target band to that of the glyceraldehyde 3-phosphate dehydrogenase band was used to measure the expression level of the proteins.

Statistical analysis

All statistical processes were performed using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). The measurement data were expressed as the mean ± standard deviation. One-way analysis of variance was adopted for intergroup comparison. P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of various concentrations of H2O2 on the cell viability of rBMSCs

As demonstrated in Fig. 1, subsequent to 6 h of H2O2 treatment, the cell viability was significantly reduced compared with the control group in a dose-dependent manner. Except 200 and 400 µM group, the reduction of cell viability in all groups had a statistical significance compared with the control group (P<0.05). In the 600 µM group, the cell survival rate approached 50% (Fig. 1A).

Figure 1 Effect of different concentrations of H2O2 and Rg1 + H2O2 treatment on the cell viability of rat bone marrow stem cells under oxidative stress. (A) H2O2 treatment (200, 400, 600 and 800 µM) for 6 h resulted in a significant reduction of cell viability compared with the control group in a dose-dependent manner. A significant reduction in cell viability was demonstrated in the treated groups, except in the 200 and 400 µM-treated groups. The cell survival rate approached 50% in the 600 µM-treated group. (B) The cell viability of H2O2 model group was significantly reduced compared with the control group (P<0.05). The cell viability significantly increased following 10 and 100 µM Rg1 treatment, compared with the H2O2 model group. There was no statistically significant difference in cell viability between the 10 and 100 µM Rg1-treated groups (P>0.05). ***P<0.05 vs. the control group and ###P<0.05 vs. the H2O2 model group. *P>0.05 vs. the control group. Data are presented as the mean ± standard deviation for three independent experiments. H2O2, hydrogen peroxide; Rg1, ginsenoside Rg1.

Effect of various concentrations of ginsenoside Rg1 on the rBMSC viability under oxidative stress

Compared with the control group, the H2O2 model group demonstrated a significant reduction in cell viability (P<0.05). The cell viability of 10 and 100 µM ginsenoside Rg1 groups increased considerably compared with the H2O2 model group with statistical significance (P<0.05). There was no significant difference in cell viability between 10 µM group and 100 µM group (P>0.05) (Fig. 1B).

Detection of apoptotic rate of rBMSCs by flow cytometry

The results of flow cytometry for each treatment indicated that the apoptosis rate of the H2O2 model group increased significantly compared with the control group (from 3.92±0.128 in the control to 59.44±3.21%; Fig. 2; P<0.05). The apoptotic ratio following H2O2 + Rg1 treatment was significantly reduced to 33.41±4.88% compared with the H2O2 model group (59.44±3.21%) and H2O2 + Rg1 +LY294002 group (49.64±2.33%; Fig. 2; P<0.05).

Figure 2 Detection of the apoptotic rate in rat bone marrow stem cells using flow cytometry. (A) Flow cytometry indicated that the apoptotic rate of the H2O2 model group increased from 3.92±0.128 to 59.44±3.21%. The apoptotic rate of the Rg1-treated group (10 µM) was significantly reduced to 33.41±4.88% compared with the H2O2 and LY294002-treated groups. (B) Histogram indicating the apoptotic rate in the control, H2O2, H2O2 + Rg1 and H2O2 + Rg1 + LY294002-treated cells. Data represent the mean ± standard error of the mean (n=3). *P<0.05 vs. the control group and #P<0.05 vs. the H2O2 model group. H2O2, hydrogen peroxide; Rg1, ginsenoside Rg1.

Detection of apoptosis of rBMSCs by TUNEL staining

Following TUNEL staining, the positive percentage of the H2O2 model group demonstrated a significant increase compared with the control group (1.88±0.133–48±4.65%; Fig. 3; P<0.05). The H2O2 + Rg1 group demonstrated a significantly decreased TUNEL-positive percentage compared with the H2O2 model group (25.29±4.33%; Fig. 3; P<0.05). The difference was also significant (P<0.05). The percentage of TUNEL-positive cells in the Akt pathway the blockage group (38.42±2.46%) increased compared with the control group (P<0.05) (Fig. 3).

Figure 3 Detection of cell apoptosis in rat bone marrow stem cells using TUNEL staining. (A) DAPI-TUNEL double staining, blue fluorescence indicates DAPI staining and green indicates TUNEL staining. (B) Quantification of apoptotic rate. The results demonstrated a significant increase in the apoptotic rate of the H2O2 model group compared with the control group (1.88±0.133–48±4.65%; P<0.05). The apoptotic rate of the Rg1-treated (10 µM) group was reduced compared with that of the H2O2 model group (25.29±4.33%; P<0.05). Compared with the Rg1-treated (10 µM) group, the apoptotic rate significantly increased in the LY294002-treated group (38.42±2.46%; P<0.05). Data are presented as the mean ± standard error of the mean (n=3). *P<0.05 vs. the control group and #P<0.05 vs. the H2O2 model group. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole; H2O2, hydrogen peroxide; Rg1, ginsenoside Rg1.

Effect of ginsenoside Rg1 on the expression levels of Bax, Bcl-2, cleaved caspase-3 and p-Akt

The results of the western blot assay indicated that the protein expression levels of cleaved caspase-3 were significantly upregulated in the H2O2 model group compared with the control group (Fig. 4; P<0.05). Increased expression of Bax and reduced expression of Bcl-2 were observed in the H2O2-treated cells compared with the control group (Fig. 4; P<0.05). Further treatment with ginsenoside Rg1 significantly prevented the upregulation of Bax and the downregulation of Bcl-2, while LY294002 treatment increased the ratio of Bax/Bcl-2, compared with the H2O2-treated group (Fig. 4; P<0.05). The expression levels of Bax and cleaved caspase-3 were significantly reduced in the ginsenoside Rg1 treatment groups compared with the H2O2 model group, while the expression levels of Bcl-2 and p-Akt were significantly upregulated (Fig. 4; P<0.05). No statistical significance was observed between the LY294002-treated and H2O2-treated groups in terms of protein expression (Fig. 4; P>0.05).

Figure 4 Detection of Bcl-2, Bax, caspase-3, p-Akt and Akt protein expression levels in H2O2-, Rg1- and LY294002-treated rat bone marrow stem cells. Western blotting was utilized to analyze (A) Bcl-2 and Bax, (B) total/cleaved caspase-3, and (C) p-Akt and Akt protein expression levels. GAPDH was used as a loading control. Results are presented as the ratio of (A) Bax to Bcl-2, (B) caspase-3 to GAPDH and (C) p-Akt to Akt. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05 vs. the control group and #P<0.05 vs. the H2O2 model group and the H2O2+Rg1+LY294002 group. p-, phosphorylated; Akt, protein kinase B; H2O2, hydrogen peroxide; Rg1, ginsenoside Rg1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Discussion

BMSCs are known as 'seeds' in cell replacement therapy and tissue engineering due to their strong proliferation and multi-directional differentiation potential. BMSCs transplantation therapy has attracted increasing attention. A previous study demonstrated that the hypoxic-ischemic tissues and high oxidative stress resulting from ischemia-reperfusion injury have an adverse impact on the survival of BMSCs in the transplantation area (23). As a result, the effect of BMSC transplantation in CRT is reduced. The marked accumulation of ROS may alter the redox state of the cells. Cells, tissues and organs undergo various injuries through the oxidation of DNA, proteins, lipids and other biomacromolecules. Excess ROS increase the permeability of the mitochondrial outer membrane, inducing the leakage of cytochrome c and apoptosis-inducing factors, thus this results in cell apoptosis. Therefore, it is necessary to reduce the oxidative stress in the transplantation region and to inhibit the apoptosis of BMSCs under oxidative stress.

Ginsenoside Rg1 is a phytoestrogen (28), exhibiting anti-oxidative and anti-apoptotic potential in myocardial cells and nerve cells (29,30). However, it remains to be reported whether ginsenoside Rg1 has an antagonist effect against the apoptosis of BMSCs. To verify the anti-oxidative effect of ginsenoside Rg1 in BMSCs, the cells were pretreated with different concentrations of ginsenoside Rg1 (1–100 µM) for 24 h prior to H2O2 treatment. The results of the CCK-8 assay indicated that ginsenoside Rg1 pretreatment significantly improved the survival of rBMSCs under oxidative stress. To further confirm the anti-apoptotic effect of ginsenoside Rg1 in BMSCs, the apoptosis of rBMSCs was determined using flow cytometry and TUNEL staining under H2O2-induced oxidative stress. The results indicated that under high oxidative stress, the pretreatment of 10 µM ginsenoside Rg1 effectively reversed the H2O2-induced apoptosis of BMSCs.

Bcl-2 is an anti-apoptotic protein that acts to prevent apoptosis by inhibiting mitochondrial depolarization (31). Bax belongs to the same family as Bcl-2 and is a pro-apoptotic protein that induces apoptosis by promoting mitochondrial depolarization (32). The initiation of apoptosis is associated with the activation of promoters and the protease cascade reaction. During the protease cascade process, caspase-3 is the primary executor of apoptosis (33,34) and the downstream effector protein of several apoptotic pathways. The present study aimed to reveal the protective mechanism of ginsenoside Rg1 against the apoptosis of BMSCs under oxidative stress, thus the expression levels of apoptosis-associated proteins were detected using western blot analysis. The results indicated that H2O2-induced oxidative stress increased the intracellular expression levels of Bax and cleaved caspase-3, while reducing the expression of Bcl-2. In addition, ginsenoside Rg1 pretreatment significantly reversed this phenomenon. This further indicates that the anti-apoptotic mechanism of ginsenoside Rg1 may be associated with the inhibition of apoptotic proteins involved in the mitochondrial pathways.

The PI3K/Akt signaling pathway is one of the most important pathways discovered to be associated with cell survival (21,35). Certain stimuli activate the pathway and promote cell survival, the activated Akt is then directly involved in the regulation of cell growth, proliferation and the cell cycle (36). It exhibits an anti-apoptotic effect by enhancing the expression levels of anti-apoptotic proteins and by inhibiting the expression levels of pro-apoptotic proteins (37). Preliminary experiments of a previous study demonstrated that ginsenoside Rg1 inhibited the apoptosis of rat chondrocytes by activating the Akt signaling pathway (30). However, it remains unclear whether BMSCs apoptosis may be inhibited by ginsenoside Rg1 activating the PI3K/Akt pathway. The results of the current study demonstrated that ginsenoside Rg1 pretreatment effectively reversed the H2O2-induced downregulation of p-Akt, significantly increased the expression of Bcl-2 and inhibited the expression of Bax and cleaved caspase-3. In order to verify the role of the PI3K/Akt pathway in the anti-apoptotic effect of ginsenoside Rg1 in BMSCs, LY294002, the specific PI3K inhibitor was administered. The results indicated that the antagonistic capacity of ginsenoside Rg1 against H2O2-induced apoptosis was inhibited. This confirmed the importance of the PI3K/Akt pathway in the protective effect of ginsenoside Rg1 against the apoptosis of BMSCs.

In addition, only the proteins associated with the mitochondrial apoptosis pathway were detected, and cell apoptosis may be additionally associated with the death receptor (36) and endoplasmic reticulum pathways (37). In conclusion, ginsenoside Rg1 was demonstrated to possess an antagonistic effect against the oxidative stress-induced apoptosis of BMSCs, and the specific mechanism may be associated with the activation of the PI3K/Akt pathway.

References