二苯乙烯苷抗氧化和抗炎作用的机制研究

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120 μmol/L的二苯乙烯苷预处理4 h，然后以1 μg/mL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，50 μmol/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-SOX^TM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。

Antioxidant and anti-inflammatory effects of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in macrophages

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 (30,60,120 μmol/L) for 4 hours,and then exposed to 1 μg/mL 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 (50 μmol/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-α and 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 MitoSOX^TM and 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-α and 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,SOD₂ and CAT. Brusatol,a specific inhibitor of Nrf2,decreased TNF-α and IL-6 production in LPS-activated macrophages. CONCLUSION: TSG attenuated LPS-induced oxidative stress and inflammation in RAW264.7 macrophages through activation of Nrf2 signal pathway. Therefore,TSG may be useful as a drug to prevent or to treat inflammation-associated diseases.