Gancaonin N from Glycyrrhiza uralensis Attenuates the Inflammatory Response by Downregulating the NF-κB/MAPK Pathway on an Acute Pneumonia In Vitro Model

Abstract

Acute pneumonia is an inflammatory disease caused by several pathogens, with symptoms such as fever and chest pain, to which children are particularly vulnerable. Gancaonin N is a prenylated isoflavone of Glycyrrhiza uralensis that has been used in the treatment of various diseases in oriental medicine. There are little data on the anti-inflammatory efficacy of Gancaonin N, and its effects and mechanisms on acute pneumonia are unknown. Therefore, this study was conducted as a preliminary analysis of the anti-inflammatory effect of Gancaonin N in lipopolysaccharide (LPS)-induced RAW264.7 cells, and to identify its preventive effect on the lung inflammatory response and the molecular mechanisms underlying it. In this study, Gancaonin N inhibited the production of NO and PGE2 in LPS-induced RAW264.7 cells and significantly reduced the expression of iNOS and COX-2 proteins at non-cytotoxic concentrations. In addition, in LPS-induced A549 cells, Gancaonin N significantly reduced the expression of COX-2 and pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Moreover, Gancaonin N reduced MAPK signaling pathway phosphorylation and NF-κB nuclear translocation. Therefore, Gancaonin N relieved the inflammatory response by inactivating the MAPK and NF-κB signaling pathways; thus, it is a potential natural anti-inflammatory agent that can be used in the treatment of acute pneumonia.

Keywords: A549 cell; Gancaonin N; Glycyrrhiza uralensis; acute pneumonia; anti-inflammation; pro-inflammatory cytokines.

Figure 1

Chemical structure of Gancaonin N (A) and its effects on RAW264.7 (B) and A549 (C) cell viability. Cells were seeded in 96-well plate and treated with Gancaonin N (5–40 μM) for 24 h. The cytotoxicity was confirmed by MTT assay. Values are represented as means ± SEM.

Figure 2

Effects of Gancaonin N on nitric oxide production (A) and protein levels of PGE2 (B), iNOS (C) and COX-2 (D) in LPS-induced RAW264.7 cells. Gancaonin N (5–40 μM) was pretreated 2 h prior to LPS-induced inflammation in RAW264.7 cells, incubated for 24 h with LPS (1 μg/mL). The protein levels of PGE2, iNOS and COX-2 were investigated by immunoblotting assay. Ratio of each protein was determined by ImageJ. Values are represented as means ± SEM. ^### p < 0.001 is significantly different from non-treated group; * p < 0.05, ** p < 0.01, *** p < 0.001 are significantly different from only LPS-treated group.

Figure 3

(A–C) Effects of Gancaonin N on pro-inflammatory cytokine mRNA expression in LPS-induced A549 cells. Gancaonin N (5–40 μM) was pretreated 2 h prior to LPS-induced inflammation in A549 cells, incubated for 24 h with LPS (5 μg/mL). The mRNA expressions of cytokine were investigated by real-time PCR analysis. Values are represented as means ± SEM. ^## p < 0.01, ^### p < 0.001 are significantly different from non-treated group; * p < 0.05, ** p < 0.01 are significantly different from only LPS-treated group.

Figure 4

Effects of Gancaonin N on protein levels of pro-inflammatory cytokines (A–C) and COX-2 (D) in LPS-induced A549 cells. Gancaonin N (5–40 μM) was pretreated 2 h prior to LPS-induced inflammation in A549 cells, incubated for 24 h with LPS (5 μg/mL). The protein level of inflammatory biomarkers was investigated by immunoblotting assay. Ratio of each protein was determined by ImageJ. Values are represented as means ± SEM. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 are significantly different from non-treated group; * p < 0.05, ** p < 0.01, *** p < 0.001 are significantly different from only LPS-treated group.

Figure 5

Effect of Gancaonin N on MAPK/NF-κB signaling pathway in LPS-induced A549 cells. Gancaonin N (5–40 μM) was pretreated 2 h prior to LPS-induced inflammation in A549 cells, incubated for 6 h with LPS (5 μg/mL). The protein levels of ERK (A), p38 (B), and NF-κB p65 (C) were investigated by immunoblotting assay. Ratio of each protein was determined by ImageJ. The expression of NF-κB p65 (D) in the nucleus was confirmed using immunofluorescence assay (magnification: 400×, scale bar:75 μm). Values are represented as means ± SEM. ^### p < 0.001 is significantly different from non-treated group; * p < 0.05, ** p < 0.01, *** p < 0.001 are significantly different from only LPS-treated group.