J Bacteriol Virol.  2018 Dec;48(4):156-165. 10.4167/jbv.2018.48.4.156.

Inhibitory Effect of Ginsenosides Rh1 and Rg2 on Oxidative Stress in LPS-Stimulated RAW 264.7 Cells

• Jin Y
• Baek N
• Back S
• Myung CS
• Heo KS

• KMID: 2452068
• DOI: http://doi.org/10.4167/jbv.2018.48.4.156

Abstract

Minor ginsenosides Rh1 and Rg2 were isolated from Korean red ginseng and reported to have various biological effects on anti-inflammatory and anti-stress activities. However, the effects of Rh1 and Rg2 on antioxidant activity and their regulatory effects on the antioxidant enzymes have not been studied. Since oxidative stress is one of the major toxic inflammatory responses stimulated by lipopolysaccharides (LPS), the present study investigated the role of minor ginsenosides Rh1 and Rg2 on antioxidant effects in LPS-treated RAW 264.7 cells. In this study, we found that treatment with ginsenosides Rh1 and Rg2 strongly inhibited LPS-stimulated intracellular ROS production in cells. Luciferase assay showed that treatment with LPS reduced antioxidant response element (ARE) encoding the pARE-luc promoter activity, while ginsenosides inhibited the pARE-luc promoter activity. Moreover, ginsenosides Rh1 and Rg2 exhibited anti-oxidative activity in LPS-induced cells by upregulating antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase. Our results suggest that minor ginsenosides Rh1 and Rg2 may be potential bio-active compounds for antioxidative effects by inhibiting the generation of ROS in RAW 264.7 cells.

Keyword

Catalase; Minor ginsenosides; Glutathione peroxidase; Lipopolysaccharids; Reactive oxygen species; Superoxide dismutase

MeSH Terms

Antioxidant Response Elements
Antioxidants
Catalase
Ginsenosides*
Glutathione Peroxidase
Lipopolysaccharides
Luciferases
Oxidative Stress*
Panax
RAW 264.7 Cells*
Reactive Oxygen Species
Superoxide Dismutase
Antioxidants
Catalase
Ginsenosides
Glutathione Peroxidase
Lipopolysaccharides
Luciferases
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Figure 1 Effect of ginsenosides Rh1 and Rg2 on cell viability. RAW 264.7 cells were treated with indicated various concentrations of Rh1 and Rg2 compounds for 24 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are the mean ± SEM of experiments in triplicate (n=3).

• 

  Figure 2 Antioxidant activities of ginsenosides Rh1 and Rg2. (A) DPPH radical scavenging activity of various concentration of ginsenosides Rh1 and Rg2. (B) ABTS radical scavenging activity of various concentration of ginsenosides Rh1 and Rg2. White bar, control; black bar, ginsenoside Rh1 and Rg2; grey bar, Vit.C (positive control). Data are the mean ± SEM of experiments in triplicate (n=3). ***p<0.001 compared with no treated sample.

• 

  Figure 3 The effect of ginsenosides Rh1 and Rg2 on intracellular ROS production. (A) RAW 264.7 cells were treated with various concentrations of the compounds for 24 h. (B) Cells were preincubated with indicated concentrations of the compounds for 24 h and then activated with 500 ng/ml LPS for 2 h. Serum-free medium containing H2DCFDA was added to cells for 30 min. 2,7-Dichlorofluorescein fluorescence was evaluated using fluorescence microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Data are the mean ± SEM of experiments in triplicate (n=3). ***p<0.001 compared with no treated sample. ###p<0.001 compared with LPS alone.

• 

  Figure 4 Effect of ginsenosides Rh1 and Rg2 on ARE promoter activity. RAW264.7 cells were transfected with ARE-luc reporter and pRL-CMV for 18 h. pRL-CMV-renilla was transfected for the internal normalization. After transfection, cells were pretreated with or without ginsenosides Rh1 and Rg2 for 1 h followed by treatment with LPS 500 ng/ml for 6 h. Cells were lysed using passive lysis buffer and luciferase activities were measured with a dual-luciferase system. Open bar, no treatment; black bar, LPS 500 ng/ml. treatment; grey bar, pretreatment with Rh1 and Rg2 followed by treatment with LPS 500 ng/ml. The relative % of the control light emission was expressed as mean ± SEM of two experiments. *p<0.05 compared with no treatment control. #p<0.05 compared with LPS alone.

• 

  Figure 5 Effect of ginsenosides Rh1 and Rg2 on the expression of anti-oxidant genes. (A) Cu/Zn SOD; (B) Mn SOD; (C) catalase (CAT); (D) Glutathione peroxidase (GPx) mRNA level in RAW 264.7 cells. Cells were treated with various concentrations of the compounds for 1 h followed by treatment with LPS for 6 h. Data are the mean ± SEM of experiments in triplicate (n=3). **p<0.01 compared with no treatment control. #p<0.05, ##p<0.01 and ###p<0.001 compared with LPS alone.

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