镇肝熄风汤含药血清激活Nrf2/ARE信号通路对6-OHDA诱导PC12细胞氧化应激的保护作用  

Protective Effects of Zhen-Gan Xi-Feng Decoction-containing Serum on 6-OHDA-induced Oxidative Stress in PC12 Cells through Nrf2-ARE Pathway Activation

作　　者：赵学梅[1] 徐天娇[2] 董妙先[2] 

机构地区：[1]齐齐哈尔医学院药学院,齐齐哈尔161006 [2]齐齐哈尔医学院医药科学研究所,齐齐哈尔161006

出　　处：《世界科学技术-中医药现代化》2017年第3期486-490,共5页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委面上项目(81373629):基于PI3K/Akt/GSK-3β信号通路的镇肝熄风法抑制帕金森病氧化应激损伤的机制研究;负责人:董妙先

摘　　要：目的:研究镇肝熄风汤含药血清对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)致PC12细胞氧化应激的保护作用及其相关机制。方法:应用镇肝熄风汤低剂量(8 g·kg^(-1))、中剂量(16 g·kg^(-1))和高剂量组(32 g·kg^(-1))大鼠的含药血清或空白血清预处理PC12细胞1 h后,加100μM 6-OHDA共孵育24 h后收集细胞。二氯荧光素双醋酸盐(DCFH-DA)荧光探针染色法结合荧光酶标仪检测活性氧(Reactive Oxygen Species,ROS)水平,采用实时定量PCR检测核转录因子E2相关因子2(Nuclear Factor-erythroid 2 Related Factor 2,Nfe2l2))基因Nfe2l2、血红素加氧酶-1(Heme Oxygenase-1,HO-1)、谷氨酸-半胱氨酸连接酶催化亚基(Glutamate-CysteineLigase Catalyticsubunit,GCLc)和调节亚基(GCLm)mRNA表达。采用荧光素酶报告基因系统检测抗氧化反应元件(Antioxidant Response Element,ARE)活性。结果:镇肝熄风汤含药血清抑制了6-OHDA诱导的氧化应激,上调了6-OHDA处理细胞中Nfe2l2、HO-1和GCLc mRNA表达,但是对GCLm mRNA表达没有显著影响。结论:镇肝熄风汤含药血清对6-OHDA诱导PC12细胞氧化应激具有保护作用,其机制可能与上调Nfe2l2 mRNA表达,使ARE激活进一步诱导其下游II相解毒酶基因和抗氧化酶基因HO-1和GCLc mRNA表达有关。This paper was aimed to study the protective effects and related mechanisms of Zhen-Gan Xi-Feng （ZGXF） decoction containing serum on 6- OHDA- induced oxidative stress in PC12 cells. The ZGXF decoction containing rat serum with low-, medium-, and high-dose （8, 16, or 32 g·kg^-1） or blank serum was used to preprocess PC12 ceils for 1 h, and cultured together with 100 p.M 6-OHDA for 24 h. And then, cells were collected. The fluorescent probe 2 ＇,7 ＇- dichlorofluorescin diacetate （DCFH-DA） and fluorescence microplate reader were used to detect the level of reactive oxygen species （ROS）. Real-time quantitative PCR was used to analyze the mRNA expressions of nuclear factor erythroid 2-related factor 2 （Nrf2）, Nfe212, heine oxygenase-1 （HO-1）, glutamate-cysteine ligase catalytic subunit （GCLc）, and GCL modulatory subunit （GCLm）. The luciferase report gene system was used to detect the antioxidant response element （ARE） activation. The results showed that ZGXF decoction-containing serum inhibited the 6-OHDA-induced oxidative stress, upregulated the Nfe212, HO-1 and GCLc mRNA expressions in cells processed with 6-OHDA. However, it has no significant effect on GCLm mRNA expression. It was concluded that ZGXF decoction-containing serum had protective effects on 6- OHDA- induced oxidative stress in PC12 cells. Its mechanism may be correlated with the upregulation on Nfe212 mRNA expression, which activated ARE and further induced its downstream gene of phase II detoxifying enzyme, as well as the HO-1 and GCLc mRNA expressions of antioxidant enzyme gene.

关 键 词：镇肝熄风汤 6-羟基多巴胺 活性氧簇 Nfe2l2 抗氧化反应元件 含药血清 

分 类 号：R285.5[医药卫生—中药学]