Abstract
BackgroundPanax 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.MethodsA 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 NO2− 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.ResultsPNFS (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.ConclusionsPNFS 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.
Highlights
Panax Notoginseng flower saponins (PNFS) are the main active component of Panax notoginseng (Burk) F
The effect of PNFS on RAW264.7 macrophage viability To evaluate the effect of PNFS on the viability of RAW264.7 macrophages, we applied various PNFS concentrations (0–800 μg/mL) and performed a Cell Counting Kit-8 (CCK-8) assay
PNFS suppresses nitric oxide (NO) overproduction in LPS-stimulated RAW264.7 macrophages The inhibitory effect of PNFS on NO overproduction in LPS-stimulated RAW264.7 macrophages was examined to evaluate the anti-inflammatory effect of PNFS
Summary
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. H. Chen flower bud (PNF) can be used to treat hypertension closely related to inflammatory response [1, 2], chemotherapy stomatitis, pharyngitis and other inflammatory diseases [3,4,5]. The methanol extract of PNF was shown to block the NFkappa B signaling pathway and alleviate the lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages [6]. Panax notoginseng flower saponins (PNFS), extracted from PNF, were reported to be the main bioactive constituent underlying PNF’s therapeutic effect [7]. The composition of PNFS is different from that of Panax notoginseng saponins (PNS), extracted from the Panax notoginseng (Burk) F. The antihypertensive effect of PNFS might be partially associated with its antiinflammatory effect
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