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

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.

2-Phenylnaphthalene Derivatives Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Mediators by Downregulating of MAPK/NF-κB Pathways in RAW 264.7 Macrophage Cells

“…In response to LPS, macrophages were activated and released pro-inflammatory mediators such as NO, iNOS, and COX-II as well as several cytokines (e.g., TNF-α and IL-6) [26]. Overexpression of either pro-inflammatory mediators or cytokines has been implicated in many inflammatory diseases such as rheumatoid arthritis, asthma, and myocarditis [3].…”

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

“…Finally, to determine why the different P. gingivalis strains induce diverse responses in host cells, inflammatory signaling pathways were measured in epi4 cells. There are three parallel MAPK signaling pathways, including the p38, ERK, and JNK signaling pathways 49 . Different microbial pathogens that infect distinct host cells may activate different signaling pathways.…”

Panax ginseng as a potential modulator of macrophages

“…These mediators are tightly regulated by host immunity. The inflammatory cytokines are one of the major LPS-induced inflammation TNF-α, IL-6, NO/iNOS, TLR4, MAPK, NF-κB [5] Igongsan NO/iNOS, NF-κB, caspase-1, COX-2 [7] Panaxynol NF-κB, Nrf2 [9] P. ginseng TNF-α, IL-1, IL-6, IL-8, IL-10, TGF-β [20] Rb1, Rb2, and Rc TNF-α, cAMP PDE [21] Rg1, Rh1, and 20(S)-protopanaxtriol TNBS-induced inflammation TLR4, NF-κB, IL-1β [4] Rb1 and compound K IL-1β, IL-6, NO/iNOS, COX-2, IRAK-1, IKK-β, NF-κB, ERK, JNK, p38 [6] Rg1 and compound K Zymosan-mediated inflammation ERK, p38 MAPK, TNF-α, IL-6, IL-12, ROS, superoxide [22] P. ginseng Infection-induced inflammation TNF-α, IL-1β, NO, PI3K/AKT [12] P. ginseng TNF-α, IL-1β, IL-6, IFN-γ, IL-12, IL-18, TLR2, MyDD88, JNK, p38, NF-κB [11] signals in cellular communication. Cells communicate with neighbor cells or transduce "danger signals" to other cells via the inflammatory cytokines or chemokines.…”

“…Thus, ginsenoside Rg1 exhibited anti-colitic effects in mice with TNBS-induced colitis. Moreover, P. notoginseng flower saponins (PNFS) are the major active components of P. notoginseng, which was demonstrated to have anti-inflammatory activity in RAW264.7 macrophages [5] . PNFS inhibited inflammation via the suppression of TLR-4, mitogen-activated protein kinases (MAPKs), NF-κB, and iNOS expressions; however, it showed no effect on phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) expression [5] .…”

“…Moreover, P. notoginseng flower saponins (PNFS) are the major active components of P. notoginseng, which was demonstrated to have anti-inflammatory activity in RAW264.7 macrophages [5] . PNFS inhibited inflammation via the suppression of TLR-4, mitogen-activated protein kinases (MAPKs), NF-κB, and iNOS expressions; however, it showed no effect on phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) expression [5] . Moreover, ginsenoside Rb1 and compound K inhibited expression of interleukin-1 receptor-associated kinase-1 (IRAK-1), inhibitor of nuclear factor kappa (IKK)-β, NF-κB, and MAP kinases (extracellular signal-regulated kinases (ERK), c-Jun amino-terminal kinases (JNK), and p-38).…”