Korean Red Ginseng saponin fraction modulates radiation effects on lipopolysaccharide-stimulated nitric oxide production in RAW264.7 macrophage cells

Abstract

Background: In previous work, we reported that Korean Red Ginseng saponin fraction (RGSF) showed anti-inflammatory activities in vitro and in vivo.

Methods: The present study investigated the radioprotective properties of RGSF by examining its effects on ionizing radiation (IR)-enhanced and lipopolysaccharide (LPS)-mediated inflammatory responses in murine macrophage cells.

Results: RGSF induced strong downregulation of IR-enhanced and LPS-induced proinflammatory responses such as nitric oxide (NO) production (Inhibitory Concentration 50 (IC50) = 5.1 ± 0.8 μM) and interleukin-1β levels. RGSF was found to exert its radioprotective effects by inhibition of a signaling cascade that activated checkpoint kinase 2-nuclear factor-κB. In addition, RGSF strongly inhibited IR-enhanced LPS-induced expression of hemoxyganase-1, implying that the latter may be a potential target of RGSF.

Conclusion: Taken together, our data suggest that RGSF can be considered and developed for use as an effective radioprotective agent with minimal adverse effects.

Keywords: 20(S)-protopanaxadiol saponins; Panax ginseng; ionizing radiation; macrophages.

Fig. 1

IR enhances LPS-induced production of NO in RAW264.7 cells. RAW264.7 cells (5 × 10⁴ cells/mL) were irradiated using the indicated doses using a blood γ irradiator and incubated at 37°C for 24 h. Irradiated cells were stimulated with 0.1 μg/mL LPS for 24 h. (A) The culture supernatant was used for NO₂⁻ determination using Griess reagent. (B) Corresponding microscopic images (200×) of RAW264.7 cells showed that irradiated cells were widened and dendrite formation was enhanced. Scale bar = 50 μm. IR, ionizing radiation; LPS, lipopolysaccharide; NO, nitric oxide. ***p < 0.001 versus LPS.

Fig. 2

Effect of RGSF on cell viability and production of NO in IR-enhanced LPS-stimulated RAW264.7 macrophages. (A) RAW264.7 cells (5 × 10⁴ cells/mL) were incubated with or without RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 10 min and irradiated (10 Gy) using a blood γ irradiator and incubated at 37°C for 24 h. Cells were washed twice with phosphate-buffered saline. Cells were incubated with or without RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 10 min and stimulated with LPS (0.1 μg/mL) for 24 h. The culture supernatant was used for NO₂^– determination using Griess reagent. Each bar graph represents mean ± standard error of the mean of at least four independent experiments. *p < 0.05, **p < 0.01 versus the IR- and LPS-treated groups. (B) Percentage inhibition of RGSF on NO production was analyzed before and after treatment with RGSF. (C) RAW264.7 cells (1 × 10⁵ cells/mL) were incubated with RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 24 hours. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed for evaluation of the cytotoxic effect of RGSF. IR, ionizing radiation; LPS, lipopolysaccharide; RGSF, red ginseng saponin fraction.

Fig. 3

Effect of RGSF on IL-1β mRNA and protein levels on IR- and/or LPS-stimulated RAW264.7 cells. RAW264.7 cells were incubated with or without RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 10 min and irradiated (10 Gy) using a blood γ irradiator and incubated at 37°C for 24 h. Cells were washed twice with phosphate-buffered saline and stimulated with LPS (0.1 μg/mL) for 24 h. mRNA and protein levels for IL-1β were evaluated by (A) semiquantitative polymerase chain reaction and (B) enzyme-linked immunosorbent assay, respectively. (A) The results shown are representative of three independent experiments and a bar graph. The graph represents the mean ± standard error of the mean of at least three independent experiments. (B) Data are presented as the mean ± SEM of at least three independent experiments. GAPDH, glyceraldehyds-3-phosphate dehydrogenase; IL, interleukin; IR, ionizing radiation; LPS, lipopolysaccharide; RGSF, red ginseng saponin fraction. *p < 0.05, **p < 0.01 compared to the IR + LPS-treated groups.

Fig. 4

Effect of RGSF on proinflammatory transcriptional factor activation. RAW264.7 cells were cotransfected with the plasmid constructs nuclear factor-κB, activator protein-1, and TK-Renilla using an electroporator. Cells were treated with the indicated concentrations of RGSF in the absence or presence of LPS (1 μg/mL) for 6 hours. Cells were then lysed and luciferase promoter activity was determined using a luminometer. Each bar graph represents mean ± standard error of the mean of at least four independent experiments. LPS, lipopolysaccharide; RGSF, red ginseng saponin fraction. *p < 0.05 versus LPS.

Fig. 5

Effect of RGSF on DNA damage related gene activation. RAW264.7 cells (1 × 10⁶ cells/mL) were incubated with or without RGSF (20 μg/mL) for 10 min and irradiated (10 Gy) using a blood gamma irradiator and incubated at 37°C for 30 min. Cells were then lysed and immunoblotting was performed. RGSF, red ginseng saponin fraction.

Fig. 6

Effect of RGSF in ionizing-radiation-enhanced HO-1 expression against LPS stimulation in RAW264.7 cells. RAW264.7 cells were incubated with or without RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 10 min and irradiated (10 Gy) using a blood γ irradiator and incubated at 37°C for 24 h. Cells were then washed twice with phosphate-buffered saline and stimulated with LPS (0.1 μg/mL) for 24 h. HO-1 expression levels were identified by immunoblotting. Data are representative of three independent experiments. HO, hemoxygenase; LPS, lipopolysaccharide; RGSF, red ginseng saponin fraction.