摘要
目的:以小鼠巨噬细胞RAW264.7细胞为研究对象,探讨二苯乙烯苷(TSG)介导的抗氧化和抗炎作用及其调控机制。方法:30、60和120μmol/L的二苯乙烯苷预处理4 h,然后以1μg/m L的脂多糖(LPS)刺激小鼠巨噬细胞(RAW264.7)12 h,并设置空白对照组、地塞米松(100 nmol/L)阳性对照和Nrf2抑制剂寒鸦子酸(brusato,50μmol/L)干预组,其中地塞米松和brusatol预处理RAW264.7细胞4 h。观察光镜下RAW264.7细胞形态学的改变并统计其被激活比例;ELISA法检测培养液中TNF-α和IL-6的水平;通过DCFH-DA和Mito-SOXTM荧光探针染色,流式细胞仪检测细胞内和线粒体内的ROS水平变化;免疫荧光法检测Nrf2的表达水平变化;Western blot检测核内Nrf2蛋白水平变化;Real-time PCR测定Nrf2下游抗氧化酶HO-1、SOD2、SOD1、CAT和GPX-1表达。结果:与空白对照组细胞相比,LPS处理后RAW264.7细胞活化,胞体增大,形成大量伪足,培养基中TNF-α和IL-6增多。二苯乙烯苷可抑制LPS诱导的RAW264.7细胞激活,降低培养基中TNF-α和IL-6,且效果优于阳性对照药物地塞米松。同时,二苯乙烯苷可促进Nrf2核转位,并促进下游抗氧化酶HO-1、SOD2和CAT表达,减轻细胞内ROS生成。而brusatol可通过阻断Nrf2活性抑制二苯乙烯苷的抗炎作用。结论:二苯乙烯苷可激活Nrf2及其下游抗氧化酶的表达,具有良好的抗炎和抗氧化作用,是一种炎症相关疾病的候选药物。
OBJECTIVE:To explore the induction of antioxidant and anti-inflammation effects of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside(TSG)in LPS-stimulated RAW264.7 macrophages.METHODS:RAW264.7 cells were pretreated with TSG(30,60,120μmol/L)for 4 hours,and then exposed to 1μg/m L LPS for 12 hours.In addition,a blank control and two positive control groups were set up.For the positive controls,cells were treated with dexamethasone(100 nmol/L)or brusatol(50μmol/L)at 4 h prior to LPS stimulation.Morphological changes were captured under a microscope,and the activated cell rate was analyzed.Concentrations of TNF-αand IL-6 in the culture medium were assessed using specific ELISA kits.Levels of superoxide anions and reactive oxygen species within the cells were detected using flow-cytometry after staining with Mito SOXTMand DCFH-DA,respectively.Moreover,immunofluorescence staining and western-blot methods were used to investigate protein expression and sub-cellular localization of Nrf2 in the cells.Real-time PCR was used to detect expression of Nrf2-related antioxidant enzymes,including HO-1,SOD2,SOD1,CAT and GPX-1.RESULTS:Compared with the control group,LPS activated macrophages exhibited elongated cell morphology with pseudopodium-like protrusions.LPS also strongly promoted the level of pro-inflammatory cytokines,including TNF-αand IL-6.However,pretreatment with TSG inhibited LPS-induced ROS production and inflammatory cytokines secretion.Interestingly,the anti-inflammatory effect of TSG was better than Dex.TSG administration promoted Nrf2 protein expression and its translocated into the nucleus.Moreover,TSG alleviated LPS-induced oxidative stress through regulating Nrf2-related antioxidant enzymes,including HO-1,SOD2and CAT.Brusatol,a specific inhibitor of Nrf2,decreased TNF-αand IL-6 production in LPS-activated macrophages.CONCLUSION:TSG attenuated LPS-induced oxidative stress and inflammation in RAW264.7macrophages through activation of Nrf2 signal pathway.Therefore,TSG may be useful as a drug to prevent or to treat inflammation-associated diseases.
作者
陈子卓
徐宇航
赵九洲
朱敬朴
郑义鹏
李文丽
吴浩芝
海春旭
于卫华
CHEN Zizhuo;XU Yuhang;ZHAO Jiuzhou;ZHU Jingpu;ZHENG Yipeng;LI Wenli;WU Haozhi;HAI Chunxu;YU Weihua(School of Basic Medical Sciences,Air Force Medical University,Xi’an 710032;Department of Toxicology,Key Lab of Hazard Assessment and Control in Special Operational Environment of Ministry of Education,Shaanxi Provincial Key Lab of Free Radical Biology and Medicine,School of Public Health,Air Force Medical University,Xi’an 710032,Shaanxi,China)
出处
《癌变·畸变·突变》
CAS
2020年第2期105-111,117,共8页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金青年科学基金(31800706)
国家自然科学基金面上项目(21677176,81473010)。
