title: 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: 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 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.
  
  
  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.
authors.affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Xiao-Xu
authors.initials: XX
authors.lastname: Peng
authors.affiliation: Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437 People's Republic of China.
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Zhang
authors.affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Xiao-Ling
authors.initials: XL
authors.lastname: Wang
authors.affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Ting-Jie
authors.initials: TJ
authors.lastname: Ye
authors.affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Hua
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Yan
authors.affiliation: School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Wei
authors.affiliation: School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Zhong-Ping
authors.initials: ZP
authors.lastname: Wu
authors.affiliation: School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Hu
authors.affiliation: School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Chun-Pu
authors.initials: CP
authors.lastname: Zou
authors.affiliation: Hypertension Laboratory, Cardiovascular Department, Long Hua Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200032 People's Republic of China.
authors.forename: You-Hua
authors.initials: YH
authors.lastname: Wang
authors.affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Hu