Preincubation with a low-dose hydrogen peroxide enhances anti-oxidative stress ability of BMSCs

Objective To investigate the effects of low-concentration hydrogen peroxide pretreatment on the anti-oxidative stress of the bone marrow mesenchymal stem cells (BMSCs). Methods Rabbit BMSCs were isolated and cultured by density gradient centrifugation combined with the adherence method. Then, the third generation of well-grown BMSCs was continuously treated with 50-μM hydrogen peroxide (H2O2) for 8 h as the optimal pretreatment concentration and the BMSCs were continuously applied for 24 h with 500 μM H2O2, and the optimal damage concentration was determined as the oxidative stress cell model. The experiment was divided into three groups: control group, high-concentration H2O2 injury group (500 μM), and low-concentration H2O2 pretreatment group (50 μM + 500 μM). In each group, the DCFH-DA fluorescence probe was used to detect the reactive oxygen species (ROS). ELISA was used to detect the activity of superoxide dismutase (SOD) and catalase (CAT), and the TBA method was used to detect malondialdehyde (MDA). The mitochondrial membrane potential was detected by JC-1. The cell viability was detected by CCK-8 method, while flow cytometry and TUNEL/DAPI double staining were performed to detect cell apoptosis. Hence, the effect of H2O2 pretreatment on the anti-oxidative stress of BMSCs was investigated. One-way analysis of variance was performed using SPSS 19.0 statistical software, and P < 0.05 was considered statistically significant. Results A large number of typical BMSCs were obtained by density gradient centrifugation and adherent culture. The oxidative stress cell model was successfully established by 500-μM H2O2. Compared with the high-concentration H2O2 injury group, the low-concentration H2O2 pretreatment reduced the production of ROS [(62.33 ± 5.05), P < 0.05], SOD and CAT activities significantly increased (P < 0.05), and MDA levels significantly decreased (P < 0.05). The mitochondrial membrane potential fluorescence changes, the ratio of red/green fluorescence intensity of the high-concentration H2O2 injury group was less, and the ratio of the low-concentration H2O2 pretreatment group was significantly higher than that. The ratio of red/green increased by about 1.8 times (P < 0.05). The cell viability and survival rate of BMSCs were significantly increased in low-concentration H2O2 pretreatment group (P < 0.05), and the cell apoptosis rate was significantly decreased (P < 0.05). Conclusion Pretreatment with low-concentration H2O2 can enhance the anti-oxidative stress ability and reduce their apoptosis of BMSCs under oxidative stress.


Background
BMSCs are pluripotent stem cells that have the potential for multi-directional differentiation [1]. In recent years, due to its high proliferative capacity and high differentiation ability, it has attracted widespread attention in stem cell research. At present, the use of BMSC transplantation for the treatment of injury has been proven to be a very promising [2][3][4]. However, clinical practice and experiments have shown that ischemia, hypoxia, and inflammatory cell infiltration in the injured area produce a large amount of ROS, forming an oxidative stress microenvironment. This results in the death of most of the transplanted BMSCs within 24 h after transplantation, badly affecting the transplantation [5]. How to improve the survival rate of cells after transplantation is an urgent problem to be solved in clinical research and application of stem cells.
Oxidative stress is the key cause of death of transplanted BMSCs [6]. The important mechanism is that the oxidative stress microenvironment in the osteonecrosis area increases the production of ROS and/or decreases the ability of scavenging ROS, resulting in an imbalance between the production and clearance of ROS [7,8]. Excessive ROS can increase mitochondrial membrane permeability, mitochondrial swelling, mitochondrial permeability (mMPTP) opening, and mitochondrial DNA (mtDNA) damage. This leads to dysfunction of mitochondrial electron transport chain, disturbance of tricarboxylic acid cycle, disturbance of ATP synthesis, decrease of transmembrane potential, release of cytochrome C, activation of Caspase-9, formation of caspasedependent apoptosis pathway, and final induction of BMSCs apoptosis [9]. Therefore, determining how to eliminate excessive ROS to enhance the survival and anti-apoptosis ability of transplanted stem cells is the key to improve the effective utilization of stem cells and enhance the therapeutic effect.
Adaptive cytoprotection refers to the application of "sub-traumatic" factors or "survival-promoting" factors to stimulate cells in advance in order to reduce the damage to cells caused by subsequent more serious damage factors [10]. Therefore, We hypothesized that oxidative stress pretreatment of BMSCs can enhance its antioxidant stress, thereby enhancing the survival and proliferation of BMSCs in the necrotic zone, thus improving the therapeutic effect of transplantation. The present study established an oxidative stress cell model by H 2 O 2 , and BMSCs were treated with the best pretreatment concentration and the best damage concentration and observed the survival of BMSCs under different concentrations of H 2 O 2 . Furthermore, the effect of low-concentration hydrogen peroxide pretreatment on the anti-oxidative stress ability of BMSCs was evaluated, providing a theoretical basis for improving the efficacy of BMSC transplantation.

Animals
In the present study, 4-6-week-old specific pathogenfree (SPF) male New Zealand white rabbits (1.0-2.0 kg) were provided by the Animal Research Center of Guizhou Medical University.

Isolation and culture of rabbit BMSCs
In the present study, 4-6-week-old New Zealand white rabbits were used. Under sterile conditions, the bone marrow fluid was taken from the distal femur and proximal humerus, and the cells were separated by density gradient centrifugation. Then, these were incubated in a cell incubator (37°C, 5% CO 2 ). When the primary cells reached 80-90% of confluence, these are passaged at a ratio of 1:3. Cells passaged up to the 3rd generation were used for the experimental treatment.

Establishment of the oxidative stress model for BMSCs
The 3rd generation BMSCs were divided into three groups: control group, high-concentration H 2 O 2 injury group (500 μM), and low-concentration H 2 O 2 pretreatment group (50 μM + 500 μM). When cell confluence reached approximately 80%, according to the experimental group, the low-concentration H 2 O 2 pretreatment group was first treated with 50 μM of H 2 O 2 for 8 h, recovered for 12 h, and finally treated with 500 μM of H 2 O 2 for 24 h, the high-concentration H 2 O 2 injury group was directly treated with 500 μM of H 2 O 2 for 24 h, and the control group was routinely cultured.

Detection of intracellular ROS
The detection of cellular ROS was carried out using the fluorescent probe DCFH-DA, which can be oxidized to the progesterone high-fluorescent compound dichlorofluorescein (DCF). Then, these cells were incubated with DCFH-DA for 30 min at 37°C and washed twice with phosphate-buffered saline (PBS). The fluorescence intensity was observed using a laser confocal microscope. Five fields were randomly selected under a high-power microscope, and the fluorescence intensity was calculated by fluorescence expression.
Evaluation of SOD, MDA, and CAT activity After cells are treated with H 2 O 2 , these cells were digested with trypsin and centrifuged at 1000 rpm for 10 min, and the cell pellet was collected. Then, these cells were disrupted by ultrasonography, centrifuge for 10 min at 12,000 rpm, and the supernatant was taken for the experiment. Each indicator was tested according to kit instructions.

Detection of cell activity by CCK-8
After cells are treated with H 2 O 2 , these cells were washed three times with PBS, and 100 μL of a complete medium and 10 μL of CCK-8 solution were added to each well. After incubation for 2 h, the enzyme-linked immunosorbent assay was performed at 450 nm. Then, the absorbance value (OD) was measured.

Annexin V-FITC/PI-detected apoptosis
The H 2 O 2 -treated cells were washed twice with PBS, and these cells were resuspended in binding buffer. Then, 100 μL of the cell suspension was transferred to a 5-ml centrifuge tube, and 5 μL of FITC and 5 μL of propidium iodide (PI) were added. Next, these cells were gently vortexed and incubated for 15 min in the dark at room temperature (25°C). Afterwards, 400 μL of binding buffer was added to each tube and analyzed by flow cytometry.

TUNEL and DAPI double staining detection of cell apoptosis
Cells were washed with PBS, fixed in 4% formaldehyde solution for 25 min at 4°C, and washed with PBS for 5 min, for three times. Then, 1% Triton X-100 was incubated for 5 min at room temperature and washed five times with PBS for three times. Afterwards, 50 μL of equilibration buffer was added to each group and equilibrated for 10 min at room temperature. Subsequently, cells were incubated with 50 μL of TdT working solution for 60 min in the dark at 37°C. Five fields were randomly selected for the high-power microscope for apoptotic cell counting, and each experiment group was repeated at least three times.
Observation of mitochondrial membrane potential by JC-1 JC-1 was prepared according to the manufacturer's instructions. Cells were incubated with the stains for 30 min at 37°C and washed three times with PBS. Finally, the fluorescence was observed under a laser confocal microscope. Five fields were randomly selected under a high-power microscope, and the red/green fluorescence intensity ratio was calculated. Each experiment group was calculated for at least three times.

Statistical methods
All data were analyzed using SPSS 19.0 statistical software. Measurement data were expressed as mean ± standard deviation ( x ± SD). One-way analysis of variance was used for multiple comparisons. The LSD t test was used for comparisons between groups. P < 0.05 was considered statistically significant.

Changes in cell morphology after H 2 O 2 induction
Cells not treated with H 2 O 2 had normal cell morphology, were spindle-shaped, and had few apoptotic cells (Fig. 1a). Cells in the high-concentration H 2 O 2 treatment group became smaller, had a long spindle shape, and the number of apoptotic cells significantly increased (Fig. 1b). However, the morphology of cells in the low-concentration H 2 O 2 pretreatment group did not significantly change, and the apoptosis was significantly reduced (Fig. 1c).

Intracellular ROS
The changes in absorption value and fluorescence intensity of each group were detected by a fluorescence microscope. The control group was not treated with H 2 O 2 , there was merely a very small amount of ROS in cells, and the content of ROS is (4.53 ± 0.49) (Fig. 2a). Compared with the control group, treatment with 500 μM of H 2 O 2 for 24 h significantly increased ROS levels, and the content of ROS was (88.3 ± 7.67), which showed strong green fluorescence (Fig. 2b). It is suggested that excessive ROS is produced after being treated with a high concentration of H 2 O 2 . However, after pretreatment with 50 μM of H 2 O 2 for 8 h, these intracellular ROS levels were effectively reduced, (62.33 ± 5.05) (Fig. 2c), and the difference was statistically significant (Fig. 2d).

SOD, MDA, CAT, and cell viability
The experimental results show that MDA in the pretreatment group was 2.83 ± 0.28 nmol/mg (Fig. 2e), while SOD, CAT, and cell viability were 34.11 ± 1.72 U/ mg, 17.59 ± 0.68 U/mg, and 77.08 ± 4.88%, respectively (Figs. 2f-h). Compared with non-pretreatment, MDA content significantly decreased, SOD, CAT, and cell viability increased. These results suggest that 50-μM H 2 O 2 pretreatment can improve the oxidative damage of cells by increasing the activities of endogenous antioxidant enzymes.

Intracellular mitochondrial membrane potential
The protective effect of low-concentration H 2 O 2 pretreatment on mitochondria could be observed by JC-1 staining. Normal cells revealed high red and high green fluorescence (Fig. 3a). However, when cells were exposed to high concentrations of H 2 O 2 (500 μM) for 24 h, Δψm rapidly depolarized, the green fluorescence increased, and the red fluorescence decreased, the ratio of red to green is 36.34 ± 5.62% (Fig. 3b). After the 50-μM H 2 O 2 pretreatment, the green fluorescence intensity was reduced and red fluorescence was restored, and the ratio of red to green is 66.52 ± 4.71% (Fig. 3c). The pretreatment group was 1.8 times higher than the nonpretreatment group (Fig. 3d). This suggests that lowconcentration H 2 O 2 pretreatment can achieve antiapoptotic effects by inhibiting the mitochondrial damage pathway.

Annexin-V/PI double staining was used to detect apoptosis
The annexin V/PI apoptosis assay revealed that the apoptotic rate was 2.56 ± 0.17% in the blank group (Fig. 4a) and 39.67 ± 3.20% in the non-pretreatment group (Fig.  4b). Compared with non-pretreatment, the apoptosis rate in the pretreatment group significantly decreased, 24.26 ± 2.10% (Fig. 4c), and the difference was statistically significant (Fig. 4d). Hence, a low-concentration H 2 O 2 pretreatment reduces apoptosis and promotes cell survival under oxidative stress.

TUNEL/DAPI detects apoptosis
The results of apoptosis detected by TUNEL/DAPI method showed that compared with the control group (Fig. 5a), the proportion of TUNEL-positive cells in the high-concentration H 2 O 2 injury group significantly increased, and the apoptosis was 25.72 ± 2.33% (Fig. 5b). However, the proportion of TUNEL-positive cells decreased in the low-concentration H 2 O 2 pretreatment group, and the apoptosis rate was 12.67 ± 0.72% (Fig. 5c). Compared with the non-pretreatment group, the apoptosis Fig. 3 Detection of mitochondrial membrane potential by JC-1. a Control group (×400); b High-concentration H 2 O 2 injury group (×400). c Lowconcentration H 2 O 2 pretreatment group (×400). d Quantitative analysis of membrane potential changes; n = 3; Compared with the control group, * P < 0.05; Compared with the high-concentration H 2 O 2 injury group, # P < 0.05 rate of the pretreatment group decreased by approximately 1/2 (Fig. 5b). These results suggest that pretreatment can significantly reduce BMSC apoptosis under oxidative stress.

Discussions
The ischemic and hypoxic microenvironment in osteonecrosis area makes cells suffer strong oxidative stress, resulting in changes and loss of cell function, inhibition of cell proliferation, and apoptosis, which seriously affects the effect of cell therapy [11,12]. Studies have shown that the oxidative stress microenvironment in the ischemic necrotic area of the femoral head causes the grafted BMSCs to suffer oxidative stress, causes a large amount of ROS, in BMSCs to induce premature senescence and apoptosis of BMSCs, and decreases the proliferation and osteogenic differentiation of surviving BMSCs, which seriously affects the repair effect of the tissue-engineered bone on ANFH [13]. Strengthening the anti-oxidant stress ability of BMSCs in the transplantation area is the key to improve the curative effect of transplantation.
Numerous studies have shown that pre-repeated short-term low-intensity damage stimulation can induce cells or the body to tolerate the subsequently sustained similar high-intensity damage stimulation, which is a ubiquitous adaptive protection phenomenon. Liu and other studies [14] have shown that after myocardial infarction in rats, the transplantation of cardiac progenitor cells (CPCs) and the H 2 O 2 pretreatment of CPCs can significantly improve the survival rate of CPCs, improve ventricular systolic function, and improve efficacy. Li and other reports [15] on diabetic cardiomyopathy, high glucose, and the H 2 O 2 pretreatment of mesenchymal stem cells (MSCs) can significantly improve the survival, proliferation, and angiogenesis of MSCs and promote cardiac repair. Therefore, in this study, we used the oxidative stress model of BMSCs induced by H 2 O 2 . BMSCs were pretreated with a low concentration of H 2 O 2 at first, and then, the effect was observed under the condition of a high concentration of H 2 O 2 . It was found that pretreatment with a low concentration of H 2 O 2 could significantly improve the resistance of BMSCs to oxidative stress.
As our experimental results show, after treating BMSCs with high concentrations of H 2 O 2 , the flow cytometry revealed that the apoptotic rate significantly increased after treatment with high concentrations of H 2 O 2, and the apoptotic rate was 39.67 ± 3.20%, and cell viability was 31.13 ± 2.11%. Furthermore, DAPI staining revealed that some of the nucleus contractions became smaller, and the nucleoplasm was densely stained or fragmented. This indicates that these cells were obviously damaged, suggesting that the oxidative stress environment was not conducive to the survival of BMSCs. However, when cells were pretreated with 50-μM lowconcentration H 2 O 2 for 8 h, cell viability significantly increased to 67.09 ± 5.38%, and the apoptosis rate was 24.26 ± 2.10% in the pretreatment group. These results show that apoptosis was significantly lower than that in the high-concentration injury group, and the cell viability rate increased significantly. This confirms that low-concentration H 2 O 2 pretreatment could significantly improve the cell survival of BMSCs after oxidative stress injury and inhibit the apoptosis.
The occurrence of apoptosis is a complex process that involves multiple factors, ROS production, mitochondrial depolarization, chromatin agglutination, and nuclear fission [16]. ROS is a general term for oxygen-containing free radicals and free radical-forming peroxides associated with oxygen metabolism in living organisms, and the oxidizing properties are very active [17]. ROS can cause membrane lipid peroxidation, protein cross-linking and degradation, deoxyribonucleic acid cleavage, and the mitochondrial dysfunction of BMSCs [18]. Under physiological conditions, the body continuously produces active oxygen, and the body's antioxidant system continuously removes active oxygen, which is in a dynamic equilibrium, and does not damage the body [19]. However, when harmful stimuli occur, a large amount of active oxygen is generated, and the antioxidant system has limited the ability to remove these, eventually leading to oxidative damage [20]. The main systems for scavenging ROS in cells are antioxidant enzymes such as SOD and CAT, the intracellular levels of both of them can represent the changes of intracellular anti-oxidant capacity to a certain extent. The present experiment revealed that pretreatment with a low concentration of H 2 O 2 can significantly reduce the production of intracellular ROS and reduce cellular oxidative stress damage. The results show that the activities of SOD and CAT in a low-concentration H 2 O 2 pretreatment group were significantly higher than those in a high-concentration H 2 O 2 injury group, and the contents of MDA and ROS decreased significantly, suggesting that H 2 O 2 pretreatment may increase the activity of SOD and CAT, so as to enhance the resistance to oxidative damage.
Mitochondria are the energy and metabolic centers of eukaryotes and are also organelles that play a key regulatory role in apoptosis and signaling [21]. The mitochondrial membrane potential declined as a preliminary event of apoptosis. Oxidative stress injury leads to apoptosis through the mitochondrial pathway, which destroys the stability of the mitochondrial membrane, causes cytochrome C to be released from the mitochondria to the cytoplasm, then binds to the apoptotic protein activator in the cytoplasm, and leads to apoptosis by proteolysis [22]. In this study, the mitochondrial membrane potential of cells was detected using JC-1 method, and damage to the mitochondrial membrane potential induced by H 2 O 2 was observed [23]. When the mitochondrial membrane potential becomes high, JC-1 forms a polymer that produces red fluorescence, while when the mitochondrial membrane potential is low, it produces a green fluorescence. This allows changes in the mitochondrial membrane potential to be detected by fluorescence color transitions [24,25]. In our results, the red fluorescence in the high-concentration H 2 O 2 injury group could be observed, the green fluorescence was significantly lower, and the ratio of red to green is 36.34 ± 5.62%, indicating that H 2 O 2 can decrease the mitochondrial membrane potential. Furthermore, the red-green ratio in the pretreatment group significantly increased, the ratio is 66.52 ± 4.71%, indicating that these cells maintained a high membrane potential level. These results show that low-concentration H 2 O 2 pretreatment can maintain the mitochondrial membrane potential level and protect mitochondrial function to some extent.
The limitations of the present study include the following, although this experimental study proved that oxidative stress preconditioning significantly enhanced the antioxidant stress of cells, but there are still many problems that have not been clarified. The mechanism of preconditioning enhancing antioxidant stress is not completely clear, which signals are regulated by preconditioning. In addition, our study only explored the enhancement of antioxidant stress by oxidative stress preconditioning in vitro. When BMSCs are used for transplantation in vivo, there are many factors affecting its survival, such as post-injury ischemia, hypoxia microenvironment, and various inflammatory factors. Therefore, on how to improve the efficiency of BMSC transplantation, there are still many problems to be studied.
In conclusion, low-concentration H 2 O 2 pretreatment can significantly increase the activity of SOD and CAT after oxidative stress injury, reduce the levels of ROS and MDA in cells, alleviate DNA and mitochondrial membrane damage, reduce cell apoptosis, and significantly promote the survival of BMSCs under oxidative stress conditions.