Protective effects of ginsenoside Rg2 against H2O2-induced injury and apoptosis in H9c2 cells

Abstract

Ginsenoside Rg2 is one of the major active components of ginseng and has many biological activities. This study aimed to investigate the protective effects of ginsenoside Rg2 against H2O2-induced injury and apoptosis in H9c2 cells. The results showed that pretreatment with ginsenoside Rg2 not only increased cell viability, but also decreased lactate dehydrogenase (LDH) release. Ginsenoside Rg2 inhibited the decrease of SOD, GSH-PX activities and the increase of MDA content induced by H2O2. Meanwhile, the levels of ROS generation and cardiomyocyte apoptosis in ginsenoside Rg2 group significantly reduced when compared with the model group. Western blot analyses demonstrated that ginsenoside Rg2 up-regulate level of Bcl-2 expression and down-regulate levels of Bax, Caspase-3, -9 expression. These findings indicated that ginsenoside Rg2 could protect H9c2 cells against H2O2-induced injury through its actions of anti-oxidant and anti-apoptosis.

Keywords: Ginsenoside Rg2; apoptosis; hydrogen peroxide; oxidative stress.

Figure 1

Chemical structure of ginsenoside Rg2.

Figure 2

Effects of ginsenoside Rg2 on cell viability. A. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, 10, 30, 100, or 300 μg/mL) for 10 h. Cell viability was measured by MTT assay. B. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. Cell viability was measured by MTT assay. Values are expressed as mean ± SD from three independent experiments. ##P < 0.01 compared with the control group; **P < 0.01 compared with the model group.

Figure 3

Morphological changes in H9c2 cells. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. Magnification: ×200.

Figure 4

Effects of ginsenoside Rg2 on LDH activity. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. Data presented are the mean ± SD. ##P < 0.01 compared with the control groups; **P < 0.01 compared with the model group.

Figure 5

Effects of ginsenoside Rg2 on ROS accumulation in H9c2 cells. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. A. ROS generation was measured by the DCF fluorescence intensity. B. Quantitative analysis of ROS generation. Data presented are the mean ± SD. ##P < 0.01 compared with the control groups; **P < 0.01 compared with the model group.

Figure 6

Effects of ginsenoside Rg2 on (A) MDA, (B) SOD and (C) GSH-PX. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. Data presented are the mean ± SD. ##P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the model group.

Figure 7

Effects of ginsenoside Rg2 on apoptosis. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. Cell apoptosis was evaluated by DAPI staining. Magnification: ×200.

Figure 8

Effects of ginsenoside Rg2 on protein levels of apoptotic-related molecules in H9c2 cells. H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2 (1, 3, or 10 μg/mL) for 4 h followed by treatment of 150 μM H₂O₂ for 6 h. Model group only treated with 150 μM H₂O₂ for 6 h. A. Representative western blots. B. Quantitative analyses for Bax. C. Quantitative analyses for Bcl-2. D. Quantitative analyses for Caspases-3. E. Quantitative analyses for Caspases-9. Data presented are the mean ± SD. ##P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the model group.