摘要
目的研究核因子E2相关因子2(Nrf2)在天麻素(GSTD)抑制油酸(OA)诱导的肝细胞氧化应激中的作用。方法体外培养HL-7702细胞,以Nrf2特异性的小干扰RNA(si RNA)转染细胞以沉默其表达,以浓度为100μg/m L的GSTD处理细胞24 h后提取细胞总蛋白以及核、质蛋白,以免疫印迹法检测Nrf2和血红素加氧酶-1(HO-1)的表达水平。在抗氧化实验中,Nrf2 si RNA转染后以浓度为0.6 mmol/L的OA与GSTD共同处理细胞24 h,以试剂盒检测细胞内超氧化物歧化酶(SOD)的活性以及活性氧簇(ROS)和丙二醛(MDA)的含量。结果与对照组细胞比较,Nrf2 si RNA转染细胞后Nrf2总蛋白、细胞核内Nrf2蛋白和HO-1蛋白的基础表达水平均显著下降(P<0.01)。与对照组细胞比较,GSTD使细胞核内Nrf2蛋白和HO-1蛋白的表达水平分别平均增加约90.2%和80.3%(P<0.01)。与单用GSTD处理的细胞比较,GSTD促进Nrf2蛋白细胞核转位和上调HO-1表达的作用能被Nrf2si RNA完全阻断(P<0.001)。与对照组细胞比较,OA处理后可诱导细胞产生氧化应激(P<0.01);与单加OA的细胞比较,GSTD与OA共同处理细胞后能使SOD的活力显著增加,ROS和MDA的水平显著下降(P<0.01);与OA+GSTD组细胞比较,GSTD的抗氧化作用能被Nrf2 si RNA完全阻断(P<0.001)。结论 Nrf2通路在GSTD抑制OA诱导的HL-7702细胞氧化应激的活性中发挥关键作用。
Objective To investigate the role of nuclear factor erythroid-2-related factor-2 (Nrf2) in the activity of gastrodin (GSTD) in suppressing oleic acid- (OA-) induced oxidative stress in hepatocytes. Methods HL-7702 cells were cultured in vitro, small interfering RNA (siRNA) specific for Nrf2 was used to transfect the cells in order to silence its expression. GSTD at a concentration of 100 μg/mL was used to treat the cells for 24 h; cell total proteins, nuclear and cytoplasmic proteins were extracted for Western blot analysis of the expression levels of Nrf2 and heme oxygenase-1 (HO-1). In the antioxidant experiments, cells were treated with 0.6 mmol/L of OA together with GSTD for 24 h after Nrf2 siRNA transfection. Intracellular superoxide dismutase (SOD) activity, reactive oxygen species (ROS) and malondialdehyde (MDA) levels were determined by kits. Results Compared with the control cells, after the transfection of Nrf2 siRNA, the cellular baseline expression levels of total Nrf2, nuclear Nrf2 and HO-1 proteins declined greatly (P 〈 0.01). Compared with the control cells, GSTD increased the nuclear Nrf2 protein and HO-1 protein levels averagely by 90.2% and 80.3%, respectively (P 〈 0.01). Compared with cells treated with GSTD alone, the stimulating activities of GSTD on the nuclear translocation of Nrf2 protein and the expression of HO-1 were totally blocked by Nrf2 siRNA (P 〈 0.001). Compared with the control cells, the ceils developed oxidative stress after the administration of OA (P 〈 0.01). Compared with cells treated with OA alone, when GSTD was co-administered with OA, the SOD activity in- creased but the levels of ROS and MDA decreased greatly in HL-7702 cells (P 〈 0.01). Compared with ceils treated with OA+GSTD, the antioxidant activity of GSTD could be blocked by Nrf2 siRNA completely (P 〈 0.001). Conclusion The Nrf2 pathway plays a critical role for the activity of GSTD in suppressing OA-induced oxidative stress in HL-7702 cells.
出处
《中国医药导报》
CAS
2016年第19期10-13,共4页
China Medical Herald
关键词
天麻素
核因子E2相关因子2
基因沉默
油酸
氧化应激
Gastrodin
Nuclear factor erythroid-2- related factor-2
Gene silencing
Oleic acid
Oxidative stress