Panax Notoginseng flower saponins (PNFS) inhibit LPS-stimulated NO overproduction and iNOS gene overexpression via the suppression of TLR4-mediated MAPK/NF-kappa B signaling pathways in RAW264.7 macrophages

摘要

Background Panax Notoginseng flower saponins (PNFS) are the main active component of Panax notoginseng (Burk) F. H. Chen flower bud (PNF) and possess significant anti-inflammatory efficacy. This study aims to explore the mechanisms underlying PNFS’ antiflammatory action in RAW264.7 macrophages. Methods A cell counting kit-8 assay was used to determine the viability of RAW264.7 macrophages. Anti-inflammation effects of PNFS in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were measured based on the detection of nitric oxide (NO) overproduction (Griess method, DAF-FM DA fluorescence assay and NO ₂? scavenging assay), and interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha gene overexpression (real-time PCR and ELISA). Inducible nitric oxide synthase (iNOS) gene overexpression was determined by real-time PCR and western blotting. iNOS enzyme activity was also assayed. The mechanisms underlying the suppression of iNOS gene overexpression by PNFS were explored using real-time PCR and western blotting to assess mRNA and protein levels of components of the Toll-like receptor 4 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-kappa B) signaling pathways. Results PNFS (50, 100, 200?μg/mL) significantly reduced LPS-induced overproduction of NO (P??0.001, P??0.001, P??0.001) and IL-6 (P?=?0.103, P??0.001, P??0.001), but did not affect TNF-alpha overproduction. PNFS (50, 100, 200?μg/mL) also markedly decreased LPS-activated iNOS (P??0.001, P??0.001, P??0.001) and TLR4 gene overexpression (P?=?0.858, P?=?0.046, P?=?0.005). Furthermore, treatment with PNFS (200?μg/mL) suppressed the phosphorylation of MAPKs including P38 (P?=?0.001), c-Jun N-terminal kinase (JNK) (P?=?0.036) and extracellular-signal regulated kinase (ERK) 1/2 (P?=?0.021). PNFS (200?μg/mL) inhibited the activation of the NF-kappa B signaling pathway by preventing the phosphorylation of inhibitor of NF-kappa B alpha (I-kappa B alpha) (P?=?0.004) and P65 (P?=?0.023), but PNFS (200?μg/mL) could not activate the LPS-induced PI3K-Akt signaling pathway. Conclusions PNFS significantly down-regulated iNOS gene overexpression and thereby decreased NO overproduction via the inhibition of TLR4-mediated MAPK/NF-kappa B signaling pathways, but not the PI3K/Akt signaling pathway.