Anti‑inflammatory effect of Chrysanthemum zawadskii, peppermint, Glycyrrhiza glabra herbal mixture in lipopolysaccharide‑stimulated RAW264.7 macrophages

Abstract

The normal inflammatory reaction protects the body from harmful external factors, whereas abnormal chronic inflammation can cause various diseases, including cancer. The purpose of the present study was to investigate the anti‑inflammatory activity of a mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra (CPG) by analyzing the expression levels of inflammatory mediators, cytokines and transcription factors in lipopolysaccharide (LPS)‑stimulated Raw264.7 cells. A nitric oxide assay, ELISA, western blotting and immunofluorescence staining were performed to investigate the anti‑inflammatory activity of the CPG mixture. Pretreatment of Raw264.7 cells with CPG inhibited the increase of inflammatory mediators (inducible nitric oxide synthase, cyclooxygenase‑2 and IFN‑β) induced by LPS. Additionally, it inhibited the production of pro‑inflammatory cytokines (TNF‑α, IL‑6 and IL‑1β). CPG suppressed LPS‑induced phosphorylation of STAT1, AKT, Iκb and NF‑κB. Furthermore, CPG inhibited the translocation of NF‑κB into the nucleus. In summary, CPG could inhibit LPS‑induced inflammation, which occurs primarily through the AKT/Iκb/NF‑κB signaling pathway in RAW264.7 cells.

Keywords: Chrysanthemum zawadskii; Glycyrrhiza glabra; NF‑κB; STAT1; anti‑inflammation; herbal mixture; peppermint.

Figure 1.

Effects of CPG on cell viability, NO levels, PGE₂ levels, iNOS and COX-2 expression in LPS-stimulated RAW264.7 macrophages. (A) RAW264.7 cells were treated with CPG at the indicated concentrations for 24 h and relative cell viability was assessed using a WST-1 assay. RAW264.7 cells were pretreated with CPG at the indicated concentrations and stimulated with LPS for 16 h. Levels of (B) NO and (C) PGE₂ in the culture supernatants were analyzed using a Griess assay and ELISA, respectively. (D) iNOS and COX-2 expression was determined by western blot analysis. (E) Relative density of iNOS was calculated using ImageJ. (F) Relative density of COX-2 was calculated using ImageJ. Data are presented as the mean ± SD. All groups labelled with the same lower case letter (a-e) were not significantly different from each other (P>0.05), whereas groups labelled with different lower case letters were significantly different (P<0.05). CPG, mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra; LPS, lipopolysaccharide; NO, nitric oxide; PGE₂, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2.

Figure 2.

Effect of CPG on TNF-α, IL-6 and IL-1β production in LPS-stimulated RAW264.7 macrophages. RAW264.7 cells were pretreated with CPG at the indicated concentrations and stimulated with LPS for 16 h. Levels of (A) TNF-α, (B) IL-6 and (C) IL-1β in the culture supernatants were analyzed using an ELISA. Data are presented as the mean ± SD. All groups labelled with the same lower case letter (a-d) were not significantly different from each other (P>0.05), whereas groups labelled with different lower case letters were significantly different (P<0.05). CPG, mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra; LPS, lipopolysaccharide.

Figure 3.

Effects of CPG on LPS-induced NF-κB activation and Akt phosphorylation in RAW264.7 macrophages. RAW264.7 cells were pretreated with CPG at the indicated concentrations and stimulated with LPS for 30 min. (A) Translocation of NF-κB p65 were determined using immunofluorescence. NF-κB (green) was detected and nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (B) DNA binding activity of NF-κB p65 were determined using ELISA. Phosphorylation levels of (C) NF-κB p65, IκB and (F) Akt were measured by western blot analysis. (D) Relative density of p-NF-κB p65 was calculated using ImageJ software. (E) Relative density of p-IκB was calculated using ImageJ software. (F) Relative density of p-AKT was calculated using ImageJ software. Data are presented as the mean ± SD. All groups labelled with the same lower case letter (a-e) were not significantly different from each other (P>0.05), whereas groups labelled with different lower case letters were significantly different (P<0.05). CPG, mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra; LPS, lipopolysaccharide; p-, phosphorylated; OD, optical density.

Figure 4.

Effects of CPG on IFN-β expression and STAT1 activation in LPS-stimulated RAW264.7 macrophages. RAW264.7 cells were pretreated with CPG at the indicated concentrations and stimulated with LPS for 16 h. (A) IFN-β expression in culture supernatant was determined using ELISA. RAW264.7 cells were pretreated with CPG at the indicated concentrations and stimulated with LPS for 3 h. (C) STAT1 phosphorylation was determined by western blot analysis. (B) Relative density was calculated using ImageJ software. Data are presented as the mean ± SD. All groups labelled with the same lower case letter (a-e) were not significantly different from each other (P>0.05), whereas groups labelled with different lower case letters were significantly different (P<0.05). CPG, mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra; LPS, lipopolysaccharide; p-, phosphorylated.

Figure 5.

Effects of CPG on HO-1 expression in LPS-stimulated RAW264.7 macrophages. RAW264.7 cells were pretreated with CPG at the indicated concentrations and stimulated with LPS for 16 h. (A) Expression levels of HO-1 were determined by western blot analysis. (B) HO-1 activity was evaluated using an ELISA. Data are presented as the mean ± SD. All groups labelled with the same lower case letter (a-e) were not significantly different from each other (P>0.05), whereas groups labelled with different lower case letters were significantly different (P<0.05). CPG, mixture of Chrysanthemum zawadskii, peppermint and Glycyrrhiza glabra; LPS, lipopolysaccharide; HO-1, heme oxygenase-1.