Artemisia iwayomogi (Dowijigi) inhibits lipopolysaccharide-induced inflammation in RAW264.7 macrophages by suppressing the NF-κB signaling pathway

Abstract

Inflammatory diseases are an important health concern and have a growing incidence worldwide. Thus, developing novel and safe drugs to treat these disorders remains an important pursuit. Artemisia iwayomogi, locally known as Dowijigi (DJ), is a perennial herb found primarily in Korea and is used to treat various diseases such as hepatitis, inflammation and immune disorders. In the present study, the anti-inflammatory effects of a polyphenolic extract from the DJ flower (PDJ) in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells were investigated. Cell cytotoxicity was assessed using the MTT assay. The production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) was measured by Griess and ELISA analysis, respectively. The expression levels of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX2) were examined by western blot analysis. Reverse transcription-quantitative PCR was performed to detect the mRNA expression levels of pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β, as well as COX2 and iNOS. The production of NO and PGE₂ was significantly decreased following treatment with PDJ. The mRNA expression levels of TNFα, IL-6, IL-1β, COX2 and iNOS were significantly decreased in LPS-induced PDJ co-treated cells compared with the group treated with LPS alone. Western blot analysis indicated that PDJ downregulated the LPS-induced expression of iNOS and COX2, as well as the expression of NF-κB proteins. In conclusion, the present study demonstrated that PDJ exerted anti-inflammatory effects in LPS-induced macrophage cells by suppressing the NF-κB signaling pathway. Therefore, PDJ may be used as a potential therapeutic agent in inflammation.

Keywords: Dowijigi; NF-κB signaling; anti-inflammation; cyclooxygenase-2; inducible nitric oxide synthase.

Figure 1.

High-performance liquid chromatography chromatogram of polyphenols obtained from the flower extract of Artemeisa iwayonogi (Dowijigi).

Figure 2.

Cytotoxic effect of PDJ on RAW264.7 cells. RAW264.7 cells were pretreated with or without LPS (1 µg/ml) at 37°C for 1 h and then subsequently treated with PDJ (0, 0.5, 1, 2.5, 5 or 10 µg/ml) at 37°C for 24 h. (A) Effect of PDJ on non-LPS-induced cell viability in RAW264.7 cells. (B) Effect of PDJ on LPS-induced cell viability in RAW264.7 cells. Data are presented as the mean ± SEM of three independent experiments. **P<0.005 vs. LPS-only treated group. PDJ, polyphenolic extract from the Dowijigi flower; LPS, lipopolysaccharide.

Figure 3.

Effect of PDJ on LPS-induced production of the inflammatory mediators NO and PGE₂ in RAW264.7 cells. RAW264.7 cells were pretreated with LPS (1 µg/ml) at 37°C for 1 h and then subsequently treated with PDJ (0, 2.5 or 5 µg/ml) for 24 h. (A) NO production. (B) PGE₂ production. Data are presented as the mean ± SEM of three independent experiments. ^###P<0.001 vs. untreated group; **P<0.005 vs. LPS-only treated group; ***P<0.001 vs. LPS-only treated group. PDJ, polyphenolic extract from the Dowijigi flower; LPS, lipopolysaccharide; NO, nitric oxide; PGE₂, prostaglandin E₂.

Figure 4.

Effect of PDJ on LPS-induced iNOS and COX2 protein expression levels in RAW264.7 cells. RAW264.7 cells were pretreated with LPS (1 µg/ml) at 37°C for 1 h and then subsequently treated with PDJ (0, 2.5 or 5 µg/ml) at 37°C for 24 h. (A) Protein expression levels of iNOS and COX2 in LPS-induced RAW264.7 cells co-treated with PDJ. β-actin was used as the loading control. (B) Relative expression of iNOS and COX2 bands from western blotting were quantified by densitometry. Data are presented as the mean ± SEM of three independent experiments. ^###P<0.001 vs. untreated group; *P<0.05 vs. LPS-only treated group; **P<0.01 vs. LPS-only treated group; ***P<0.001 vs. LPS-only treated group. PDJ, polyphenolic extract from the Dowijigi flower; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; COX2, cyclooxygenase-2.

Figure 5.

Effect of PDJ on the LPS-induced mRNA expression of COX2 and iNOS in RAW264.7 cells. RAW264.7 cells were pretreated with LPS (1 µg/ml) at 37°C for 1 h and then subsequently treated with PDJ (0, 2.5 or 5 µg/ml) at 37°C for 24 h. mRNA expression levels of (A) COX2 and (B) iNOS were measured by reverse transcription-quantitative PCR. Data are presented as the mean ± SEM of three independent experiments. ^###P<0.001 vs. untreated group; ***P<0.001 vs. LPS-only treated group. PDJ, polyphenolic extract from the Dowijigi flower; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; COX2, cyclooxygenase-2.

Figure 6.

Effect of PDJ on the LPS-induced pro-inflammatory cytokine expression levels in RAW264.7 cells. RAW264.7 cells were pretreated with LPS (1 µg/ml) at 37°C for 1 h and then subsequently treated with PDJ (0, 2.5 or 5 µg/ml) for at 37°C 24 h. mRNA expression levels of the pro-inflammatory cytokines (A) IL-1β, (B) IL-6 and (C) TNFα were measured by reverse transcription-quantitative PCR. Data are presented as the mean ± SEM of three independent experiments. ^###P<0.001 vs. untreated group; *P<0.05 vs. LPS-only treated group; **P<0.01 vs. LPS-only treated group; ***P<0.001 vs. LPS-only treated group. PDJ, polyphenolic extract from the Dowijigi flower; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor.

Figure 7.

Effect of PDJ on LPS-induced protein expression of p-p65, p65, p-IκBα and IκBα. RAW264.7 cells were pretreated with LPS (1 µg/ml) at 37°C for 1 h and then subsequently treated with PDJ (0, 2.5 or 5 µg/ml) for at 37°C 24 h. β-actin was used as the loading control. (A) Western blot analysis and (B) the relative expression of p-p65/p65 and p-IκBα/IκBα ratios quantified by densitometry. Data are presented as the mean ± SEM of three independent experiments. ^##P<0.01 vs. untreated group; *P<0.05 vs. LPS-only treated group; **P<0.01 vs. LPS-only treated group; ***P<0.001 vs. LPS-only treated group. PDJ, polyphenolic extract from the Dowijigi flower; LPS, lipopolysaccharide; p, phosphorylated; IκBα, inhibitor of κBα.

Figure 8.

Schematic representation of the NF-κB-mediated inhibition of inflammatory responses by the polyphenolic extract of Dowijigi flower. LPS, lipopolysaccharide; TLR, Toll-like receptor; IκB, inhibitor of κB; IκBα, inhibitor of κBα; COX2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; IL, interleukin; TNF, tumor necrosis factor; NO, nitric oxide; PGE₂, prostaglandin E₂; p, phosphorylated.