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.
Keywords: Bone marrow mesenchymal stem cells; Hydrogen peroxide, Apoptosis; Oxidative stress injury; Pretreatment.