Nutr Res Pract.  2015 Dec;9(6):579-585. 10.4162/nrp.2015.9.6.579.

Sonchus asper extract inhibits LPS-induced oxidative stress and pro-inflammatory cytokine production in RAW264.7 macrophages

• Wang L
• Xu ML
• Liu J
• Wang Y
• Hu JH
• Wang MH

• KMID: 2313880
• DOI: http://doi.org/10.4162/nrp.2015.9.6.579

Abstract

BACKGROUND/OBJECTIVES
Sonchus asper is used extensively as an herbal anti-inflammatory for treatment of bronchitis, asthma, wounds, burns, and cough; however, further investigation is needed in order to understand the underlying mechanism. To determine its mechanism of action, we examined the effects of an ethyl acetate fraction (EAF) of S. asper on nitric oxide (NO) production and prostaglandin-E2 levels in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.
MATERIALS/METHODS
An in vitro culture of RAW264.7 macrophages was treated with LPS to induce inflammation.
RESULTS
Treatment with EAF resulted in significant suppression of oxidative stress in RAW264.7 macrophages as demonstrated by increased endogenous superoxide dismutase (SOD) activity and intracellular glutathione levels, decreased generation of reactive oxygen species and lipid peroxidation, and restoration of the mitochondrial membrane potential. To confirm its anti-inflammatory effects, analysis of expression of inducible NO synthase, cyclooxygenase-2, tumor necrosis factor-alpha, and the anti-inflammatory cytokines IL-1beta and IL-6 was performed using semi-quantitative RT-PCR. EAF treatment resulted in significantly reduced dose-dependent expression of all of these factors, and enhanced expression of the antioxidants MnSOD and heme oxygenase-1. In addition, HPLC fingerprint results suggest that rutin, caffeic acid, and quercetin may be the active ingredients in EAF.
CONCLUSIONS
Taken together, findings of this study imply that the anti-inflammatory effect of EAF on LPS-stimulated RAW264.7 cells is mediated by suppression of oxidative stress.

Keyword

anti-inflammatory; NO production; oxidative stress; Sonchus asper

MeSH Terms

Antioxidants
Asthma
Bronchitis
Burns
Chromatography, High Pressure Liquid
Cough
Cyclooxygenase 2
Cytokines
Dermatoglyphics
Glutathione
Heme Oxygenase-1
Inflammation
Interleukin-6
Lipid Peroxidation
Macrophages*
Membrane Potential, Mitochondrial
Nitric Oxide
Nitric Oxide Synthase
Oxidative Stress*
Quercetin
Reactive Oxygen Species
Rutin
Sonchus*
Superoxide Dismutase
Tumor Necrosis Factor-alpha
Wounds and Injuries
Antioxidants
Cyclooxygenase 2
Cytokines
Glutathione
Heme Oxygenase-1
Interleukin-6
Nitric Oxide
Nitric Oxide Synthase
Quercetin
Reactive Oxygen Species
Rutin
Superoxide Dismutase
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effect of different concentrations of EAF of S. asper on RAW264.7 macrophage viability was measured by MTT reduction assay. Data are expressed as the mean ± SD of three independent experiments.

• 

  Fig. 2 Flow cytometric analysis of intracellular accumulation of ROS after EAF treatment. The number meaning is the ROS fluorescence events after different treatment. Data are expressed as the mean ± SD of three independent experiments. (A) control (without LPS), (B) LPS, (C) 100 µg/mL with LPS, (D) 50 µg/mL with LPS (E) 25 µg/mLwith LPS, (F) 12.5 µg/mL with LPS.

• 

  Fig. 3 Effect of EAF of S. asper on the loss of mitochondrial membrane potential of LPS-stimulated macrophages. Data are expressed as the mean ± SD of three independent experiments. Lowercase letters indicate significant differences according to ANOVA test (a < b < c < d) (P < 0.05).

• 

  Fig. 4 Effect of EAF of S. asper on LPS-induced NO production in RAW264.7 macrophages. Data are expressed as the mean ± SD of three independent experiments. Lowercase letters indicate significant differences according to ANOVA test (a < b < c < d) (P < 0.05).

• 

  Fig. 5 Effect of EAF of S. asper on LPS-induced PGE2 production in RAW264.7 macrophages. Data are expressed as the mean ± SD of three independent experiments. Lowercase letters indicate significant differences according to ANOVA test (a < b < c < d) (P < 0.05).

• 

  Fig. 6 Effects of EAF of S. asper on pro-inflammtory gene expression analyzed by semi-quantitative RT-PCR. (A) EtBr staining of PCR products using primers specific for iNOS, COX-2, IL-1β, IL-6, TNF-α, and GAPDH, respectively. (B) Quantification of PCR products (A) relative to GAPDH following treatment with EAF. Data are mean ± SD of three independent experiments. Lowercase letters indicate significant differences according to ANOVA test (a < b < c < d) (P < 0.05).

• 

  Fig. 7 Effects of EAF of S. asper on antioxidant gene expression analyzed by semi-quantitative RT-PCR. (A) EtBr staining of PCR products using primers specific for MnSOD, HO-1, and GAPDH, respectively. (B) Quantification of PCR products (A) relative to GAPDH following treatment with EAF. Data are expressed as mean ± SD of three independent experiments. Lowercase letters indicate significant differences according to ANOVA test (a < b < c < d) (P < 0.05).

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