Ginsenoside Rb1 attenuates oxygen-glucose deprivation-induced apoptosis in SH-SY5Y cells via protection of mitochondria and inhibition of AIF and cytochrome c release

Abstract

To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

Figure 1

MTT assay of cellular viability. Pretreatment of GRb1 at the concentration of 1.0 µmol/L and 10.0 µmol/L significantly suppressed cellular death caused by OGD at 24h. However, 100 µmol/L GRb1 did not show protection on cellular viability. * p < 0.01 versus control group; ^# p < 0.01 versus OGD group.

Figure 2

Apoptosis detected by TUNEL staining and flow cytometry. (A) representative image of TUNEL staining; (B) statistical results; (C) flow cytometry. The cells with positive reaction to TUNEL staining showed green color and represented apoptotic cells. * p < 0.01 versus control group; ^& p < 0.05 versus OGD group; ^# p < 0.01 versus OGD group. Scale bar: 40 µm.

Figure 3

Measurement of ROS level. (A) representative images under fluorescence microscope; (B) fluorescence density statistics. Fluorescence microscopy showed the fluorescence in the OGD group was significantly higher than that in the control group, but it was suppressed by treatment with either 1.0 µmol/L or 10.0 µmol/L GRb1. Further study showed that the fluorescence density in the OGD group was 4.32 ± 0.76 times as high as that in the control group. By contrast, it was reduced markedly to 2.64 ± 0.31 and 1.5 ± 0.22 times when the cells were treated with 1.0 µmol/L or 10.0 µmol/L GRb1, respectively. This result indicated that GRb1 mitigated the increase of intracellular ROS level caused by OGD. * p < 0.01 versus control group; ^# p < 0.01 versus OGD group. Scale bar: 50 µm.

Figure 4

Measurement of mitochondrial membrane potential by flow cytometry. Treatment with 1.0 µmol/L or 10.0 µmol/L GRb1 counteracted OGD-induced reduction of mitochondrial membrane potential in SH-SY5Y cells. This result indicated that GRb1 protected mitochondrial damage caused by OGD.

Figure 5

Western blotting analysis of the content of AIF, cyto c, Bax and Bcl-2 within mitochondria, nucleus and cytoplasm. (A) representative image of western blotting; (B) statistical analysis. * p < 0.01 versus control group; ^& p < 0.05 versus OGD group; ^# p < 0.01 versus OGD group. This result indicated that the attenuation of GRb1 on the release of AIF and cytochrome c from mitochondria might be via modulating mitochondrial level of Bax and Bcl-2.

Figure 6

Immunocytochemical analysis of AIF redistribution. Immunocytochemistry showed that part of mitochondrial AIF translocated from mitochondria to nucleus in the OGD treated SH-SY5Y cells, when compared with that in the control group. However, this translocation was suppressed effectively by GRb1 either at the concentration of 1.0 µmol/L or 10.0 µmol/L. Scale bar: 10 µm.