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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.

Peng XX, Zhang SH, Wang XL, Ye TJ, Li H, Yan XF, Wei L, Wu ZP, Hu J, Zou CP, Wang YH, Hu XD - Chin Med (2015)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of PNFS on RAW264.7 macrophage viability. Data were expressed as the mean (SD) of 3 independent experiments. One-Way ANOVA test was used to analyzed the data and the result was F = 87.693; P < 0.001. Then, data were counted by SNK and LSD multiple comparisons to determine the statistical difference between two groups. The P values represented the statistical differences between each group and negative control group (without PNFS treated). *** means P <0.001
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Fig1: Effect of PNFS on RAW264.7 macrophage viability. Data were expressed as the mean (SD) of 3 independent experiments. One-Way ANOVA test was used to analyzed the data and the result was F = 87.693; P < 0.001. Then, data were counted by SNK and LSD multiple comparisons to determine the statistical difference between two groups. The P values represented the statistical differences between each group and negative control group (without PNFS treated). *** means P <0.001

Mentions: 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 CCK-8 assay. PNFS had an obvious cytotoxic effect at 400 μg/mL (P < 0.001) and 800 μg/mL (P < 0.001), but had no cytotoxic effect at 200 μg/mL and lower concentrations on RAW264.7 macrophages (Fig. 1). So we chose the concentrations at 50, 100, 200 μg/mL for further exploring the anti-inflammatory mechanisms of PNFS.Fig. 1


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.

Peng XX, Zhang SH, Wang XL, Ye TJ, Li H, Yan XF, Wei L, Wu ZP, Hu J, Zou CP, Wang YH, Hu XD - Chin Med (2015)

Effect of PNFS on RAW264.7 macrophage viability. Data were expressed as the mean (SD) of 3 independent experiments. One-Way ANOVA test was used to analyzed the data and the result was F = 87.693; P < 0.001. Then, data were counted by SNK and LSD multiple comparisons to determine the statistical difference between two groups. The P values represented the statistical differences between each group and negative control group (without PNFS treated). *** means P <0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493833&req=5

Fig1: Effect of PNFS on RAW264.7 macrophage viability. Data were expressed as the mean (SD) of 3 independent experiments. One-Way ANOVA test was used to analyzed the data and the result was F = 87.693; P < 0.001. Then, data were counted by SNK and LSD multiple comparisons to determine the statistical difference between two groups. The P values represented the statistical differences between each group and negative control group (without PNFS treated). *** means P <0.001
Mentions: 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 CCK-8 assay. PNFS had an obvious cytotoxic effect at 400 μg/mL (P < 0.001) and 800 μg/mL (P < 0.001), but had no cytotoxic effect at 200 μg/mL and lower concentrations on RAW264.7 macrophages (Fig. 1). So we chose the concentrations at 50, 100, 200 μg/mL for further exploring the anti-inflammatory mechanisms of PNFS.Fig. 1

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203 People's Republic of China.

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.

No MeSH data available.


Related in: MedlinePlus