Total flavonoids from Saussurea involucrata attenuate inflammation in lipopolysaccharide-stimulated RAW264.7 macrophages via modulating p65, c-Jun, and IRF3 signaling pathways

Abstract

Objective: To investigate the anti-inflammatory effects of the total flavonoids from Saussurea involucrata on lipopolysaccharides (LPS)-stimulated murine RAW264.7 macrophages and explore its underlying mechanism of action. Methods: Total flavonoids from Saussurea involucrata were extracted using chromatographic column method. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The production of nitric oxide was detected by Griess assay and the release of cytokines (IL-10 and TNF-alpha) and chemokines (MCP-1, MIP-1a, and CCL5/RANTES) was determined by ELISA to evaluate the anti-inflammatory activity of total flavonoids from Saussurea involucrata. Moreover, nuclear translocation of p65, c-Jun, and IRF3 was detected by immunofluorescence microscopy and Western blotting analysis was performed to determine the expression of related proteins. Results: Total flavonoids extracted from Saussurea involucrata were 751.5 mg/g and the content of rutin was 506.5 mg/g. The production of inflammatory mediators including nitric oxide, cytokines, and chemokines was effectively inhibited by total flavonoids from Saussurea involucrata. Meanwhile, total flavonoids also suppressed the nuclear translocation of p65, c-Jun, and IRF3 in LPS-stimulated RAW264.7 cells. The LPS-induced expression of iNOS and COX-2 was remarkably reduced by treatment with total flavonoids from Saussurea involucrata. Moreover, total flavonoids decreased the expression levels of p-IKKa/beta, p-TBK1, p-p38, p-ERK, p-JNK, p-p65, p-c-Jun, and p-IRF3 in LPS-exposed RAW264.7 macrophages. Conclusions: Total flavonoids from Saussurea involucrata potentially inhibit the secretion of pro-inflammatory mediators, which may be related to inhibition of p65, c-Jun, and IRF3 signaling pathways in LPS-stimulated RAW264.7 cells.