Korean J Physiol Pharmacol.  2021 Sep;25(5):395-401. 10.4196/kjpp.2021.25.5.395.

Formosanin C attenuates lipopolysaccharide-induced inflammation through nuclear factor-κB inhibition in macrophages

• Yin L
• Shi C
• Zhang Z
• Wang W
• Li M

• KMID: 2519402
• DOI: http://doi.org/10.4196/kjpp.2021.25.5.395

Abstract

Extended inflammation and cytokine production pathogenically contribute to a number of inflammatory disorders. Formosanin C (FC) is the major diosgenin saponin  found in herb Paris formosana Hayata (Liliaceae), which has been shown to exert anti-cancer and immunomodulatory functions. In this study, we aimed to investigate anti-inflammatory activity of FC and the underlying molecular mechanism. RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) or pretreated with FC prior to being stimulated with LPS. Thereafter, the macrophages were subjected to analysis of the expression levels of pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, as well as two relevant enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). The analysis revealed that FC administration blunted LPS-induced production of NO and PGE in a dose-dependent manner, while the expression of iNOS and COX-2 at both mRNA and protein levels was inhibited in LPS-stimulated macrophages pre-treated with FC. Moreover, LPS stimulation upregulated mRNA expression and medium release of TNF-α, IL-1β, and IL-6, whereas this effect was blocked upon FC pre-administration. Mechanistic studies showed that inhibitory effects of FC on LPS-induced inflammation were associated with a downregulation of IκB kinase, IκB, and p65/NF-κB pathway. Taken together, these data suggest that FC possesses an inflammation-suppressing activity, thus being a potential agent for the treatment of inflammation-associated disorders.

Keyword

Formosanin C; Inflammation; Lipopolysaccharide; Macrophage; NF-kappa B

Figure

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  Fig. 1 Cytotoxicity analysis of FC in macrophages. The cytotoxicity of FC in RAW264.7 macrophages was measured by CCK-8 assay. The macrophages were treated with various concentrations of FC (0, 0.625, 1.25, 2.5, 5, 10, and 20 μM) for 24 h. n = 3; data were presented as means ± SD. FC, formosanin C; CCK, cell counting kit-8. *Indicates p < 0.05 vs. the control sample.

• 

  Fig. 2 Effects of FC on LPS-elicited NO production and iNOS expression. (A) NO production was examined by Griess assay. (B) The mRNA expression of iNOS was evaluated by qPCR. (C) The protein expression of iNOS was assessed by Western blotting. RAW264.7 macrophages were treated with FC for 2 h and then subjected to an stimulation with 1 μg/ml LPS for 22 h. n = 3; data were presented as means ± SD. FC, formosanin C; LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric oxide synthase. *Indicates p < 0.05 vs. the control group; #indicates p < 0.05 vs. the LPS alone group.

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  Fig. 3 Effects of FC on LPS-elicited PGE production and COX-2 expression. (A) PGE production was examined by ELISA assay. (B) The mRNA levels of COX-2 was assessed by qPCR. (C) The protein levels of COX-2 was determined using Western blot analysis. RAW264.7 macrophages were pretreated with FC for 2 h and then subjected to an stimulation with 1 μg/ml LPS for 22 h. n = 3; data were presented as means ± SD. FC, formosanin C; LPS, lipopolysaccharide; PGE, prostaglandin E2; COX-2, cyclooxygenase-2. *Indicates p < 0.05 vs. the control group; #indicates p < 0.05 vs. the LPS alone group.

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  Fig. 4 Effects of FC on LPS-elicited releases of pro-inflammatory cytokines. (A–C) The mRNA expression of IL-1β (A), IL-6 (B) and TNF-α (C) was evaluated by qPCR. (D–F) The production of IL-1β (D), IL-6 (E) and TNF-α. (F) was assessed by ELISA assays. RAW264.7 macrophages were pretreated with FC for 2 h and then subjected to an stimulation with 1 μg/ml LPS for 22 h. n = 3; data were presented as means ± SD. FC, formosanin C; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β. *Indicates p < 0.05 vs. the control group; #indicates p < 0.05 vs. the LPS alone group.

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  Fig. 5 FC inhibited the activation of NF-κB pathway. (A) The phosphorylation levels of IKK, IκBα and p65 were assessed by Western blot analysis. (B) The fluorescence microscopy imaging was taken. Blue and green indicate nuclei and p65, respectively; scale bar, 50 μm. RAW264.7 macrophages were pretreated with FC for 2 h and then subjected to an stimulation with 1 μg/ml LPS for 18 h. n = 3; data were presented as means ± SD. FC, formosanin C; NF-κB, nuclear factor-kappa B; IKK, IκB kinase; LPS, lipopolysaccharide. *Indicates p < 0.05 vs. the control group; #indicates p < 0.05 vs. the LPS alone group.

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