Phyllolobium chinense Fisch Flavonoids (PCFF) Suppresses the M1 Polarization of LPS-Stimulated RAW264.7 Macrophages by Inhibiting NF-κB/iNOS Signaling Pathway

Abstract

Background: M1 macrophage plays an important role in inflammatory reaction. In this study, potential anti-inflammatory effect of Phyllolobium chinense Fisch flavonoids (PCFF) was assessed via Zebrafish acute inflammation model in vivo and LPS-induced pro-inflammatory M1 macrophage model in vitro.

Methods: The quality control of P. chinense Fisch flavonoids (PCFF) was analyzed by HPLC. Anti-inflammatory effect of PCFF on the acute injured zebrafish was evaluated by the migration of fluorescence labeled macrophages and neutrophils, and the gene expression of inflammatory factors. In addition, the anti-inflammatory mechanism of PCFF was investigated by the related gene expression and related signaling pathway regulation of pro-inflammatory mediators in LPS-induced pro-inflammatory M1 RAW264.7 macrophage.

Results: P. chinense Fisch flavonoids (PCFF) markedly suppressed macrophage and neutrophil migration and iNOS gene expression in acute injured zebrafish with tail-cutting. PCFF significantly inhibited NO overproduction and iNOS gene overexpression in LPS-sitimulated pro-inflammatory M1 RAW264.7 macrophages. What's more, PCFF could evidently decrease p65 protein production, but had no effect on the production of P38, JNK and ERK1/2 proteins.

Conclusion: P. chinense Fisch flavonoids (PCFF) have a remarkable inhibitory effect on the inflammatory response in acute injured zebrafish and LPS-stimulated M1 RAW264.7 macrophage. The pharmacological mechanism may be related to the regulation of NO overproduction and the inhibition of NF-κB/iNOS signaling pathway.

Keywords: Phyllolobium chinense Fisch flavonoids (PCFF); RAW264.7 macrophages; inducible nitric oxide synthases; inflammatory response; nuclear factor-κB signaling pathway; zebrafish.

Figure 1

High Performance Liquid Chromatography of PCFF. (A) complanatosides B reference substance; (B) complanatosides A reference substance; (C) Sample.

Figure 2

Survival rate of zebrafish. Each value indicates the mean ± SD from three independent experiments. Compared with normal group, ^*P < 0.05, ^**P < 0.01.

Figure 3

Inhibitory effect of PCFF on migration of zebrafish macrophages and neutrophils. Each value indicates the mean ± SD from three independent experiments. Compared with normal group, ^##P < 0.01; compared with control group, ^**P < 0.01. NC group untreated; Model group cut tail; PCFF group added different dose PCFF after tail cut.

Figure 4

Inhibitory effect of PCFF on expression of iNOS、TNF-α and IL-1β mRNA. Each value indicates the mean ± SD from three independent experiments. Compared with normal group, ^##P < 0.01; compared with control group, ^*P < 0.05, ^**P < 0.01. NC group untreated; Model group cut tail; PCFF group added different dose PCFF after cut tail.

Figure 5

Cytotoxicity of PCFF on RAW264.7 cells and effects of PCFF on LPS-induced NO production in RAW264.7 cells. (A) Each value indicates the mean ± SD from three independent experiments. Compared with untreated group, ^**P <0.01; (B) Compared with NC group, ^##P < 0.01; compared with LPS group, ^**P <0.01.

Figure 6

Effect of PCFF on LPS-induced inflammatory factor iNOS gene in RAW264.7 cells. Each value indicates the mean ± SD from three independent experiments. Compared with NC group, ^##P <0.01; compared with LPS group, ^**P <0.01.

Figure 7

Effects of PCFF on TNF-alpha secretion in LPS-induced RAW264.7 cells. Each value indicates the mean ± SD from three independent experiments. Compared with NC group, ^##P < 0.01.

Figure 8

Effects of PCFF on NF-κB and MAPK signaling pathway proteins in LPS-stimulated RAW264.7 macrophages. (A) Effect of PCFF on p65 and iNOS proteins; (B) Effect of PCFF on JNK, P38 and iNOS proteins; (C) Effect of PCFF on ERK1/2 and iNOS proteins. Each value indicates the mean ± SD from three independent experiments. Compared with NC group, ^##P < 0.01; compared with LPS group, ^**P < 0.01.