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

Xiao-Xu Peng; Shu-Hui Zhang; Xiao-Ling Wang; Ting-Jie Ye; Hua Li; Xiao-Feng Yan; Li Wei; Zhong-Ping Wu; Jing Hu; Chun-Pu Zou; You-Hua Wang; Xu-Dong Hu

doi:10.1186/s13020-015-0045-x

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

Chin Med. 2015 Jul 1:10:15. doi: 10.1186/s13020-015-0045-x. eCollection 2015.

Authors

Xiao-Xu Peng^#¹, Shu-Hui Zhang^#², Xiao-Ling Wang¹, Ting-Jie Ye¹, Hua Li¹, Xiao-Feng Yan¹, Li Wei³, Zhong-Ping Wu⁴, Jing Hu⁴, Chun-Pu Zou⁴, You-Hua Wang⁵, Xu-Dong Hu¹

Affiliations

¹ Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
² Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437 People's Republic of China.
³ School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
⁴ School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.
⁵ Hypertension Laboratory, Cardiovascular Department, Long Hua Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200032 People's Republic of China.

^# Contributed equally.

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.