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

Abstract

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.

Fig. 1

Observation of cell morphology using an inverted phase-contrast microscope. a Control group (×100); b High-concentration H₂O₂ injury group (×100); c Low-concentration H₂O₂ pretreatment group (×100)

Fig. 2

Detection of intracellular ROS, MDA, SOD, CAT, and cell viability. a–c Detection of ROS, control group, high-concentration H₂O₂ injury group and Low-concentration H₂O₂ pretreatment group respectively (×100); d Quantitative analysis of DCF fluorescence intensity; e Intracellular MDA content; f Intracellular SOD viability; g Intracellular CAT viability; h Cell viability; n = 5; Compared with the control group, ^*P < 0.05; Compared with the high-concentration H₂O₂ injury group, ^#P < 0.05

Fig. 3

Detection of mitochondrial membrane potential by JC-1. a Control group (×400); b High-concentration H₂O₂ injury group (×400). c Low-concentration H₂O₂ 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 H₂O₂ injury group, ^#P < 0.05

Fig. 4

Apoptosis detected by annexin-V/PI staining. a Control group (×400); b High-concentration H₂O₂ injury group (×400); c Low-concentration H₂O₂ pretreatment group (×400); d Quantitative analysis of cell apoptosis; n = 3; Compared with the control group, ^*P < 0.05; Compared with the high-concentration H₂O₂ injury group, ^#P < 0.05

Fig. 5

Apoptosis detected by TUNEL/DAPI staining. a Control group (×400); b High-concentration H₂O₂ injury group (×400); c Low-concentration H₂O₂ pretreatment group (×400); d Quantitative analysis of cell apoptosis; n = 3; Compared with the control group, ^*P < 0.05; Compared with the high-concentration H₂O₂ injury group, ^#P < 0.05