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Anti-inflammatory Effects of Schisandra chinensis (Turcz.) Baill Fruit Through the Inactivation of Nuclear Factor-κB and Mitogen-activated Protein Kinases Signaling Pathways in Lipopolysaccharide-stimulated Murine Macrophages.

Kang YS, Han MH, Hong SH, Park C, Hwang HJ, Kim BW, Kyoung KH, Choi YW, Kim CM, Choi YH - J Cancer Prev (2014)

Bottom Line: This study examined the anti-inflammatory effects of Schisandrae Fructus ethanol extract (SF) on the production of pro-inflammatory substances in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects.Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) by reducing inhibitory-κB degradation, and reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), implying that SF regulated LPS-induced NF-κB-dependent inflammatory pathways through suppression of MAPKs activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Korea.

ABSTRACT

Background: Schisandrae Fructus, the dried fruit of Schisandra chinensis (Turcz.) Baill. (Magnoliaceae), is widely used in traditional medicine for the treatment of a number of chronic inflammatory diseases. This study examined the anti-inflammatory effects of Schisandrae Fructus ethanol extract (SF) on the production of pro-inflammatory substances in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Methods: To measure the effects of SF on pro-inflammatory mediator and inflammatory cytokine's expression and production in RAW 264.7 cells, we used the following methods: cell viability assay, Griess reagent assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, Western blotting analysis and immunofluorescence staining.

Results: Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects. SF also inhibited the expression of inducible NO synthase, TNF-α, and IL-1β protein and their mRNAs in LPS-stimulated RAW 264.7 cells. Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) by reducing inhibitory-κB degradation, and reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), implying that SF regulated LPS-induced NF-κB-dependent inflammatory pathways through suppression of MAPKs activation.

Conclusions: SF may be useful for the treatment of various inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus

Inhibition of nitric oxide (NO) production by Schisandrae Fructus ethanol extract (SF) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Cells were pretreated with 500 μg/mL SF for 1 hour before incubation with LPS (100 ng/mL) for 24 hours. (A) Nitrite content was measured using the Griess reaction in culture media using a commercial enzyme-linked immunospecific assay kit. Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments (*P < 0.05 compared with control group; #P < 0.05 compared with LPS-treated group). (B) Total RNA was isolated after a 6 hours LPS treatment and reverse-transcribed using inducible NO synthase (iNOS) primers. The resulting cDNAs were then subjected to polymerase chain reaction (PCR). The reaction products were run on 1% agarose gels and visualized by ethidium bromide staining. (C) The cells were sampled and lysed following a 24 hours treatment, and equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Western blotting was performed using anti-iNOS antibody and an an enhanced chemiluminescence detection system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls for the reverse transcriptase- PCR and Western blot assays, respectively. The relative amounts of iNOS protein and mRNA were normalized with GAPDH and actin, respectively.
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f2-jcp-19-279: Inhibition of nitric oxide (NO) production by Schisandrae Fructus ethanol extract (SF) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Cells were pretreated with 500 μg/mL SF for 1 hour before incubation with LPS (100 ng/mL) for 24 hours. (A) Nitrite content was measured using the Griess reaction in culture media using a commercial enzyme-linked immunospecific assay kit. Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments (*P < 0.05 compared with control group; #P < 0.05 compared with LPS-treated group). (B) Total RNA was isolated after a 6 hours LPS treatment and reverse-transcribed using inducible NO synthase (iNOS) primers. The resulting cDNAs were then subjected to polymerase chain reaction (PCR). The reaction products were run on 1% agarose gels and visualized by ethidium bromide staining. (C) The cells were sampled and lysed following a 24 hours treatment, and equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Western blotting was performed using anti-iNOS antibody and an an enhanced chemiluminescence detection system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls for the reverse transcriptase- PCR and Western blot assays, respectively. The relative amounts of iNOS protein and mRNA were normalized with GAPDH and actin, respectively.

Mentions: The possibility that SF has anti-inflammatory properties was investigated by determining the effects of SF on the level of NO in the culture media of RAW 264.7 cells after a 24 hours treatment with 100 ng/mL LPS and SF. Treatment of RAW 264.7 cells with LPS resulted in a dramatic increase in NO production. Treatment with 500 μg/mL SF significantly inhibited this production of NO (Fig. 2A). The possibility that this inhibitory effect of SF on NO production occurred via inhibition of corresponding gene expression was investigated by determining the mRNA and protein expressions of iNOS by reverse transcriptase (RT)-PCR and Western blot analyses. Figure 2B and 2C show that mRNA and protein expression of iNOS was undetectable in RAW 264.7 cells without LPS stimulation. Treatment with LPS alone markedly increased iNOS mRNA and protein levels, while pretreatment with SF significantly suppressed these levels. The reduced expressions of iNOS mRNA and protein were consistent with the reductions in NO production in the culture media.


Anti-inflammatory Effects of Schisandra chinensis (Turcz.) Baill Fruit Through the Inactivation of Nuclear Factor-κB and Mitogen-activated Protein Kinases Signaling Pathways in Lipopolysaccharide-stimulated Murine Macrophages.

Kang YS, Han MH, Hong SH, Park C, Hwang HJ, Kim BW, Kyoung KH, Choi YW, Kim CM, Choi YH - J Cancer Prev (2014)

Inhibition of nitric oxide (NO) production by Schisandrae Fructus ethanol extract (SF) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Cells were pretreated with 500 μg/mL SF for 1 hour before incubation with LPS (100 ng/mL) for 24 hours. (A) Nitrite content was measured using the Griess reaction in culture media using a commercial enzyme-linked immunospecific assay kit. Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments (*P < 0.05 compared with control group; #P < 0.05 compared with LPS-treated group). (B) Total RNA was isolated after a 6 hours LPS treatment and reverse-transcribed using inducible NO synthase (iNOS) primers. The resulting cDNAs were then subjected to polymerase chain reaction (PCR). The reaction products were run on 1% agarose gels and visualized by ethidium bromide staining. (C) The cells were sampled and lysed following a 24 hours treatment, and equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Western blotting was performed using anti-iNOS antibody and an an enhanced chemiluminescence detection system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls for the reverse transcriptase- PCR and Western blot assays, respectively. The relative amounts of iNOS protein and mRNA were normalized with GAPDH and actin, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4285959&req=5

f2-jcp-19-279: Inhibition of nitric oxide (NO) production by Schisandrae Fructus ethanol extract (SF) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Cells were pretreated with 500 μg/mL SF for 1 hour before incubation with LPS (100 ng/mL) for 24 hours. (A) Nitrite content was measured using the Griess reaction in culture media using a commercial enzyme-linked immunospecific assay kit. Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments (*P < 0.05 compared with control group; #P < 0.05 compared with LPS-treated group). (B) Total RNA was isolated after a 6 hours LPS treatment and reverse-transcribed using inducible NO synthase (iNOS) primers. The resulting cDNAs were then subjected to polymerase chain reaction (PCR). The reaction products were run on 1% agarose gels and visualized by ethidium bromide staining. (C) The cells were sampled and lysed following a 24 hours treatment, and equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Western blotting was performed using anti-iNOS antibody and an an enhanced chemiluminescence detection system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls for the reverse transcriptase- PCR and Western blot assays, respectively. The relative amounts of iNOS protein and mRNA were normalized with GAPDH and actin, respectively.
Mentions: The possibility that SF has anti-inflammatory properties was investigated by determining the effects of SF on the level of NO in the culture media of RAW 264.7 cells after a 24 hours treatment with 100 ng/mL LPS and SF. Treatment of RAW 264.7 cells with LPS resulted in a dramatic increase in NO production. Treatment with 500 μg/mL SF significantly inhibited this production of NO (Fig. 2A). The possibility that this inhibitory effect of SF on NO production occurred via inhibition of corresponding gene expression was investigated by determining the mRNA and protein expressions of iNOS by reverse transcriptase (RT)-PCR and Western blot analyses. Figure 2B and 2C show that mRNA and protein expression of iNOS was undetectable in RAW 264.7 cells without LPS stimulation. Treatment with LPS alone markedly increased iNOS mRNA and protein levels, while pretreatment with SF significantly suppressed these levels. The reduced expressions of iNOS mRNA and protein were consistent with the reductions in NO production in the culture media.

Bottom Line: This study examined the anti-inflammatory effects of Schisandrae Fructus ethanol extract (SF) on the production of pro-inflammatory substances in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects.Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) by reducing inhibitory-κB degradation, and reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), implying that SF regulated LPS-induced NF-κB-dependent inflammatory pathways through suppression of MAPKs activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Korea.

ABSTRACT

Background: Schisandrae Fructus, the dried fruit of Schisandra chinensis (Turcz.) Baill. (Magnoliaceae), is widely used in traditional medicine for the treatment of a number of chronic inflammatory diseases. This study examined the anti-inflammatory effects of Schisandrae Fructus ethanol extract (SF) on the production of pro-inflammatory substances in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Methods: To measure the effects of SF on pro-inflammatory mediator and inflammatory cytokine's expression and production in RAW 264.7 cells, we used the following methods: cell viability assay, Griess reagent assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, Western blotting analysis and immunofluorescence staining.

Results: Stimulation of the RAW 264.7 cells with LPS caused an elevated production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin (IL)-1β, which was markedly inhibited by the pretreatment with SF without causing any cytotoxic effects. SF also inhibited the expression of inducible NO synthase, TNF-α, and IL-1β protein and their mRNAs in LPS-stimulated RAW 264.7 cells. Furthermore, SF attenuated LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) by reducing inhibitory-κB degradation, and reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), implying that SF regulated LPS-induced NF-κB-dependent inflammatory pathways through suppression of MAPKs activation.

Conclusions: SF may be useful for the treatment of various inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus