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 effec...
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-$1{\beta}$ 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.
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-$1{\beta}$ 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.
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문제 정의
The aim of this study was to determine whether EAF has anti-inflammatory effects in LPS-stimulated RAW macrophages and whether these effects are mediated by inhibition of oxidative stress. Therefore, NO release and mRNA levels of cytokines were evaluated.
asper has been used extensively as an herbal antiinflammatory for treatment of bronchitis, asthma, wounds, burns, and cough, however, further investigation is needed in order to understand the underlying mechanism. The purpose of this study was to examine the question of whether the antiinflammatory effect of S. asper was mediated by suppression of oxidative stress in RAW264.7 macrophages.
가설 설정
In this study, we examined the capacity of EAF to block NO and PGE2 production. EAF-induced expression of pro-inflammatory cytokines was associated with a reduction of oxidative stress in LPS-activated macrophages.
대상 데이터
The primers (Bioneer, Seoul, Korea) for MnSOD and HO-1 were: Forward 5'-GTGACTT- TGGGTCTTTTGAG-3', Reverse, 5'- GCTAACATTCTCCCAGTTGA-3' and Forward 5'-AAGATTGCCCAG AAAGCCCTGGAC-3', Reverse, 5'-AACTGTCGCCACCAG- AAAGCT GAG-3', respectively.
데이터처리
Values with the same letter are not significantly different by a one-way ANOVA test (P > 0.05).
이론/모형
7 mouse leukaemic monocyte macrophage cell line was obtained from the American Type Cell Culture (Rockville, MD, USA) and cultured in RPMI-1640 medium (Gibco, MD, USA) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. The cytotoxicity and growthinhibitory effect of EAF was assessed using the standard 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTT) cell viability assay following the methods of Wang et al. [15].
The effect of EAF (100, 50, 25, and 12.5 μg/mL) on viability of un-stimulated RAW264.7 cells was tested using the MTT assay.
성능/효과
After further fractionation by n-hexane, dichloromethane, ethyl acetate, and n-butanol in a stepwise manner, the yield of the ethyl acetate fraction (EAF) was 9.3 mg; this was used for the anti-inflammatory assays in this study because we found that the ethyl acetate fraction showed the highest NO inhibition rate (93.36%) at 100 μg/mL; the NO inhibition rates of the other extracts were 24.94%, 66.80%, 46.04%, and 41.10% at 100 μg/mL, respectively (data not shown in manuscript).
The anti-inflammatory effects of EAF were confirmed by its enhancement of SOD activity and intracellular GSH level in LPSactivated macrophages. In addition, results of RT-PCR showed that EAF induced up-regulation of the antioxidant genes MnSOD and HO-1 in LPS-activated macrophages. Because the antioxidant enzyme SOD and the major endogenous antioxidant GSH are major contributors to intracellular redox balance, we conclude that EAF is a strong antioxidant that protects against oxidative stress in LPS-activated macrophages.
LPS stimulation decreased SOD activity by 31% in RAW264.7 cells, compared with the control, but after treatment with 100, 50, 25, and 12.5 μg/mL EAF for 24 h, SOD activity was restored by 27%, 21%, 18%, and 7%, respectively (Table 1).
Treatment with 100, 50, 25, and 12.5 μg/mL EAF resulted in reduced LPS-induced ROS generation by 58%, 54%, 47%, and 7%, respectively.
후속연구
, singlet oxygen, thus increasing resistance to lipid peroxidation; these known compounds may contribute to the anti-inflammatory effect to some extent. However, as they compose a small proportion of EAF, conduct of further studies will be required for identification of the remaining functional components.
asper was shown to prevent CCl4-induced nephrotoxicity in rats [14]. S. asper has been used extensively as an herbal antiinflammatory for treatment of bronchitis, asthma, wounds, burns, and cough, however, further investigation is needed in order to understand the underlying mechanism. The purpose of this study was to examine the question of whether the antiinflammatory effect of S.
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