白藜芦醇经NRF2/ARE通路减轻MPTP毒性研究

目的 观察白藜芦醇(resveratrol,trans-3,4′,5trihydroxystilbene,RE)对帕金森病(PD)斑马鱼多巴胺神经元的保护作用及其机制.方法 本研究分为5组:1)空白对照组(con组);2)200μM/L MPTP处理组(MPTP组);3)60μM/L RE处理组(RE组);4)先加60μM/L RE,12 h后加200μM/L MPTP组(MR1组);5)先加200μM/L MPTP,12 h后加60μM/L RE组(M1R组),每组含斑马鱼25条.每组斑马鱼发育至第4天时观察药物对多巴胺神经元、PD相关基因和NRF2/ARE信号通路的影响.解剖显微镜观察斑马鱼的运动功能、外部形态;激光共聚焦显微镜观察多巴胺神经元;荧光定量PCR检测PD相关基因parkin、pink1、dj-1以及抗氧化相关基因sod、cat的表达;Western blot检测Nrf2蛋白表达.结果 与con组相比,MPTP组斑马鱼出现尾巴后弯,多巴胺神经元减少,RE回救多巴胺神经元数量;MPTP组PD相关基因和氧化应激相关基因表达下调,Nrf2蛋白表达水平下降;MIR与MR1组PD相关基因、氧化应激相关基因及Nrf2蛋白的表达得到援救.结论 MPTP对斑马鱼多巴胺神经元具有毒性作用,RE可减弱MPTP的毒性,可通过抗氧化应激来实现.

A Study on the Attenuation of MPTP Toxicity by Resveratrol via the NRF2/ARE Pathway

Objective To investigate the neuroprotective effect of resveratrol (trans-3,4′,5-trihydroxystilbene, RE)on dopaminergic neurons in the zebrafish infected with Parkinson′s disease (PD)and its mechanism.Methods The zebrafishes were divided into 5 groups including the blank control group,the MPTP group with 200μmol/L MPTP,the RE group with 60 um/L resveratrol,the MR1 group with 60 um/L resveratrol first and 200μmol/L MPTP second,and the M1R group with 200μmol/L MPTP first and 60 um/L resveratrol second,and each group consisted of 12 zebrafishes.The effect of drugs on the dopaminergic neurons,PD-related genes and NRF2/ARE signaling pathways was observed in each group on the fourth day.The motor function and morphology of the zebrafish were observed by microscope,and the dopaminergic neurons were observed by confocal laser scanning microscope.The expressions of PD-related genes including parkin,pink1 ,dj-1 and the antioxidant-related genes including sod and cat were detected by fluorescence quantitative polymerase chain reaction (PCR ), and the expression of nrf2 was detected by Western blotting.Results Compared with the blank control group,the zebrafish in the MPTP group had tail dorsal horn and decreased dopaminergic neurons,while the amount of dopaminergic neurons was increased by resveratrol.The expressions of PD-related genes and the antioxidant-related genes were down-regulated and the expression level of nrf2 was decreased in the MPTP group.The PD-related genes and the antioxidant-related genes in the MIR and MR1 groups were protected.Conclusion MPTP has toxic effect on dopaminergic neurons of zebrafish and resveratrol can attenuate the toxicity of MPTP via NRF2/ARE pathways.