Nutr Res Pract.  2020 Oct;14(5):453-462. 10.4162/nrp.2020.14.5.453.

Immunomodulatory effects of fermented Platycodon grandiflorum extract through NF-κB signaling in RAW 264.7 cells

• Park EJ
• Lee HJ

• KMID: 2506883
• DOI: http://doi.org/10.4162/nrp.2020.14.5.453

Abstract

BACKGROUND/OBJECTIVES
Platycodon grandiflorum (PG), an oriental herbal medicine, has been known to improve liver function, and has both anti-inflammatory and antimicrobial properties. However, little is known about the immune-enhancing effects of PG and its mechanism. In this study, we aimed to investigate whether fermented PG extract (FPGE), which has increased platycodin D content, activates the immune response in a murine macrophage cell line, RAW 264.7.
MATERIALS/METHODS
Cell viability was determined by Cell Counting Kit-8 assay and the nitric oxide (NO) levels were measured using Griess reagent. Cytokine messenger RNA levels of were monitored by quantitative reverse transcription polymerase chain reaction. To investigate the molecular mechanisms underlying immunomodulatory actions of FPGE in RAW 264.7 cells, we have conducted luciferase reporter gene assay and western blotting.
RESULTS
We found that FPGE treatment induced macrophage cell proliferation in a dosedependent manner. FPGE also modulated the expression of NO and pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The activation and phosphorylation levels of nuclear factor kappa B (NF-κB) were increased by FPGE treatment. Moreover, 5-aminoimidazole-4-carboxamide ribonucleotide, an activator of AMP-activated kinase (AMPK), significantly reduced both lipopolysaccharides- and FPGE-induced NF-κB reporter gene activity.
CONCLUSIONS
Taken together, our findings suggest that FPGE may be a novel immuneenhancing agent acting via AMPK-NF-κB signaling pathway.

Keyword

Platycodon grandiflorum; nitric oxide; interleukin-1 beta; interleukin-6; NF-kappa B

Figure

• 

  Fig. 1 FPGE induces cell proliferation in RAW 264.7 cells. The RAW 264.7 cells were treated with various concentrations of FPGE for 48 h prior to measure cell viability. Cell viability is expressed as a relative value to that of the vehicle-treated cells, which is set to 100%. The data represents the mean ± SEM (n = 3).FPGE, fermented Platycodon grandiflorum extract; VC, vehicle control.**P < 0.01, ***P < 0.001.

• 

  Fig. 2 FPGE enhances NO production in RAW 264.7 cells. Cells were treated with various concentrations of FPGE or LPS (1 μg/mL) for 24 h prior to measure nitrite level. The data were expressed as the mean ± SEM (n = 3).NO, nitric oxide; VC, vehicle control; FPGE, fermented Platycodon grandiflorum extract; LPS, lipopolysaccharides.***P < 0.001 as compared with VC.

• 

  Fig. 3 FPGE increases the mRNA level of pro-inflammatory cytokines in RAW 264.7 cells. RAW 264.7 cells were treated with FPGE (20, 100, 500 μg/mL) or LPS (1 μg/mL) for 24 h and assessed using real-time RT-PCR for (A) Tnf-α, (B) IL-1b, (C) IL-6. All gene expression values were normalized to those of Actb (β-actin) and were expressed as a relative value to that of the VC, which is set to 1. The figures show the mean ± SEM (n = 3).Tnf-α, tumor necrosis factor-α; mRNA, messenger RNA; VC, vehicle control; LPS, lipopolysaccharides; FPGE, fermented Platycodon grandiflorum extract; IL, interleukin; RT-PCR, reverse transcription polymerase chain reaction.*P < 0.05, **P < 0.01, ***P < 0.001.

• 

  Fig. 4 FPGE induces NF-κB activation in RAW 264.7 cells. (A) The cells were co-transfected with 3×κB-Luc and pNL1.1.TK constructs and treated with FPGE (20, 100, 500 μg/mL) or LPS (1 μg/mL) for 24 h prior to measure luciferase activities. Luciferase activity is expressed as a relative value to that of the VC, which is set to 100%. The figures show the mean ± SEM (n = 3). (B) The cells were treated with FPGE (20, 100, 500 μg/mL) or LPS (1 μg/mL) for 1 h prior to western blot analysis. Western blots are representative of 3 independent experiments.VC, vehicle control; FPGE, fermented Platycodon grandiflorum extract; LPS, lipopolysaccharides; pNF-κB, phospho-nuclear factor kappa B; NF-κB, nuclear factor kappa B; pIκBα, phospho-inhibitor of kappa B; IκBα, inhibitor of kappa B.**P < 0.01.

• 

  Fig. 5 FPGE-induced NF-κB activation is associated with AMPK in RAW 264.7 cells. (A) The cells were treated with FPGE (20, 100, 500 μg/mL) or LPS (1 μg/mL) or 1 h prior to western blot analysis. The phosphorylated/total ratios of (B) AMPK and (C) ACC were plotted based on the quantification of the signal intensities. (D) The cells were co-transfected with 3×κB-Luc and pNL1.1.TK constructs and treated with FPGE (500 μg/mL), LPS (1 μg/mL), compound C (10 μM), and/or AICAR (1 mM) for 24 h prior to measure luciferase activities. Luciferase activity is expressed as a relative value to that of the VC, which is set to 100%. The figures show the results from 3 independent experiments which were expressed as the mean ± SEM.FPGE, fermented Platycodon grandiflorum extract; VC, vehicle control; LPS, lipopolysaccharides; pAMPK, phospho-5′ adenosine monophosphate-activated protein kinase; AMPK, 5′ adenosine monophosphate-activated protein kinase; pACC, phospho-acetyl-CoA carboxylase; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide.*P <0.05, ***P < 0.001 as compared with VC.

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