中文摘要：

目的探讨1,5-二咖啡酰奎宁酸（1,5-diCQA）是否通过激活Nrf2而减轻1-甲基-4-苯基吡啶离子（MPP＋）所致的PC12细胞损伤,并对可能的信号通路进行研究。方法采用MPP＋处理具有多巴胺（DA）能神经元特性的PC12细胞作为帕金森病（PD）的体外模型。实验分为正常对照组、MPP＋处理组、1,5-diCQA预处理＋MPP＋处理组,为了观察1,5-diCQA预处理的浓度-效应关系,将1,5-diCQA的浓度设为10、20、50、100μmol/L。用CCK8法测定各组细胞存活率,酶标仪检测细胞谷胱甘肽（GSH）水平和活性氧族（ROS）水平,Western blot检测各组细胞的Nrf2蛋白水平。进一步采用siRNA转染沉默Nrf2,加入MAPK家族一系列激酶抑制剂后再检测上述相关指标。结果 MPP＋（250mmol/L）处理PC12细胞24h后,与正常对照组相比,细胞存活率下降,GSH耗竭,ROS生成增多,说明PD细胞模型建立成功。不同浓度的1,5-diCQA预处理可以减轻MPP＋导致的细胞损伤,且在一定范围内具有量效关系;不同浓度的1,5-diCQA预处理可以明显上调Nrf2蛋白的水平。沉默Nrf2后,1,5-diCQA预处理对MPP＋诱导损伤的PC12细胞的保护作用消失,细胞存活率未能回升[MPP＋组vs.MPP＋＋1,5-diCQA组：（19.47±1.65）%vs.（21.13±2.85）%,P=0.352 6],GSH水平在Nrf2敲除的PC12细胞中明显下降[NT siRNA组vs.Nrf2siRNA组：（15.05±1.71）nmol/mgprotein vs.（4.31±0.83）nmol/mg protein,P〈0.001],且1,5-diCQA预处理对GSH耗竭的逆转效应也消失。对MAPK激酶家族的一系列激酶进行信号通路筛选发现,Erk激酶参与了1,5-diCQA通过激活Nrf2而减轻MPP＋诱导的PC12细胞损伤这一过程。结论 1,5-diCQA可浓度依赖性地减轻MPP＋诱导的PC12氧化应激损伤。这种保护作用是通过激活Erk激酶,进而激活Nrf2,然后上调细胞内源性抗氧化系统来实现的。

英文摘要：

Objective To explore the protective mechanisms of l,5-dicaffeoylquinic acid（1,5-diCQA）preconditioning on MPP＋-induced injury on PC12 cells.Methods Rat pheochromocytoma（PC12）cells were treated with MPP＋to establish a cell model of Parkinson’s disease and then randomly divided into 3groups：1,5-diCQA pretreatment＋MPP＋group,MPP＋group and control group.The viability of cells was determined by CCK-8assay.Cellular glutathione（GSH）and reactive oxygen species（ROS）levels were measured by spectrophotometry.The expression of Nrf2 protein was detected by Western blot.SiRNA transfection was performed to silence Nrf2.After addition of a series of MAPK family kinase inhibitors,the indexes mentioned above were detected again.Results Twenty-four h after MPP＋treatment,the cell viability was significantly decreased,the GSH level depleted and ROS markedly generated,which suggested the successful establishment of the PD cell model of Parkinson’s disease.Pretreatment with 1,5-diCQA could alleviate the MPP＋-induced cell damage and the effect was in a dose-dependant manner.The protective effect of 1,5-diCQA pretreatment was abolished in Nrf2-knockdown PC12 cells;the cell viability failed to increase in MPP＋＋1,5-diCQA group[MPP＋group vs.MPP＋＋1,5-diCQA group,（19.47 ± 1.65）% vs.（21.13 ± 2.85）%,P=0.352 6];the GSH level was significantly decreased in Nrf2-knockdown PC12 cells[negative siRNA group vs.Nrf2siRNA group,（15.05±1.71）nmol/mg protein vs.（4.31±0.83）nmol/mg protein,P〈0.01];the reverse effect of 1,5-diCQA pretreatment on GSH depletion was blocked in Nrf2-knockdown PC12 cells.The Erk kinase signaling was found to be involved in the protective effect of 1,5-diCQA against the MPP ＋-induced cell damage.Conclusion 1,5-diCQA may dose-dependently alleviate the MPP ＋-induced oxidative stress injury by activating ERK signaling pathway and upregulating Nrf2 in the PC12 cells.