戊二酸致原代培养纹状体神经元神经毒性的体外研究

In Vitro Study of Glutaric Acid-Induced Neurotoxicity in Primary Cultured Striatal Neurons

目的探讨戊二酸（GA）干预体外培养纹状体神经元致神经元损伤的浓度和时间及其损伤机制。方法体外培养新生大鼠纹状体神经元,于体外培养7 d行神经元特异性烯醇化酶（NSE）免疫细胞化学染色鉴定神经元纯度。纯度达90%以上,用不同浓度GA（1~50 mmo·lL-1）孵育体外培养10 d的纹状体神经元24~96 h。倒置显微镜及赫斯特荧光染料33342（-/＋）荧光活细胞染色后荧光显微镜观察神经元形态变化;四甲基偶氮唑盐微量酶反应比色（MTT）法检测细胞线粒体功能变化;膜联蛋白-V碘/化丙啶双染流式细胞仪检测判断GA诱导纹状体神经元凋亡率及死亡率;实时定量反转录（RT）-PCR及Western blot法检测半胱氨酸天冬氨酸酶（Caspase）-3、Caspase-9表达。结果 GA处理组细胞线粒体功能受损程度呈浓度及时间依赖性改变：随着GA浓度升高,干预时间延长,神经元损伤加重。与正常对照组比较,10 mmo·lL-1、25 mmo·lL-1、50 mmo·lL-1组GA干预24~72 h纹状体神经元活力均比正常对照组显著增高（P〈0.05）;1 mmo·lL-1组干预72 h、96 h与正常对照组比较均明显升高（Pa〈0.01）。50 mmo·lL-1组孵育72 h较孵育48 h细胞凋亡显著增加[（87.63±9.17）%vs（40.90±4.10）%,P〈0.01];25 mmo·lL-1组孵育72 h凋亡率为（24.73±2.95）%。10 mmo·lL-1、25 mmo·lL-1、50 mmo.lL-1GA作用神经元1 h、6 h,Caspase-9和Caspase-3 mRNA表达明显增加,25 mmo·lL-1、50 mmo·lL-1GA处理24 h后神经元Caspase-9、Caspase-3蛋白活化片段增加。结论 GA致纹状体神经元损伤呈浓度-时间依赖性,通过Caspase-9/Caspase-3途径诱导纹状体神经元凋亡,该机制可能与GA血症Ⅰ型大鼠纹状体退行性变有关。

Objective To investigate the concentration-and time-dependent toxicity of glutaric acid（GA） on primary cultured neonatal rat striatal neurons and the possible mechanism.Methods The purity of neuronal cells from neonatal rat was determined by neuron-specific enolase（NSE） immunoreactivity,when cultured for 7 days in vitro.The cells,at least 90% pure,cultured for 10 days,were submitted to GA at concentration ranging from 1 to 50 mmol·L-1 for 24-96 hours incubation.To evaluate the severity of neuron injury,the changes of morphology were observed by inverted microscopy,whereas the nuclear morphology was observed under fluorescence microscope after an incubation of Hoechst 33342.The mitochondrial function was measured by 3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-diphenytetrazoliu-mromide method.The rates of neuronal apoptosis and necrosis were assessed via annexin-V and propidium iodide staining by flow cytometer system.The expressions of Caspase-3 and Caspase-9 were detected by quantitative reverse transcriptase-PCR and Western blot.Results Under the inverted microscopy,neurons showed obvious toxic damage in GA treatment group.Compared with control group,the mitochondrial activity was significantly decreased in a concentration-and time-dependent manner.Compared with the control group,GA group（10,25,50 mmol·L-1） significantly induced mitochondrial activity（P0.05） for 24 h,48 h,72 h,whereas at 1 mmol·L-1 concentration,an incubation of 72 h and 96 h made a significant decrease in the mitochondrial activity（Pa0.01）.The rates of neuronal apoptosis obviously increased：Having incubated for 48 h,72 h with 50 mmol·L-1 GA,the rates of apoptosis were（40.90±4.10）% and（87.63±9.17）%,respectively,and 25 mmol·L-1 GA incubated for 72 h,it was（24.73±2.95）%.The expressions of Caspase-9 and Caspase-3 mRNA were significantly up-regulated at 1 h and 6 h after 10 mmol·L-1,25 mmol·L-1 and 50 mmol·L-1 GA treatment.As well,Caspase-9 and Caspase-3 were markedly up-regulated in active protein level after 24 h treatment of 25 mmol·L-1,50 mmol·L-1 GA.Conclusions GA induced the primary rat striatal neuron damage in a concentration-and time-dependent fashion.GA initiates the apoptotic cascade in the primary cultured striatal neurons,which may be through Caspase-9/Caspase-3 pathway.This mechanism may contribute to the striatal degeneration observed in glutaric acidemia type Ⅰ.