N-乙酰半胱氨酸拮抗鱼藤酮致PC12细胞凋亡的研究

Preventive Effect of N-acetylcysteine against Rotenone-induced Apoptosis in PC12 Cells

目的探讨N-乙酰半胱氨酸(NAC)对PC12细胞的保护作用及机制。方法采用鱼藤酮(1μmol/L)处理PC12细胞24 h,建立帕金森病细胞模型;在该模型中,N-乙酰半胱氨酸(500μmol/L)预处理PC12细胞30 min。采用AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡率,以双氢溴化乙啶(DHE)和双氢罗丹明123(DHR123)为染料,采用流式细胞术检测细胞内活性氧水平;比色法检测还原型谷胱甘肽(GSH)水平,同时检测细胞Caspase-3酶活性和线粒体膜电位。结果 1μmol/L鱼藤酮处理PC12细胞后,细胞凋亡率为42.0%,DHE和DHR123荧光强度分别为正常组的230%和333%,GSH含量为101 nmol/mg prot,细胞线粒体膜电位为正常组的46%,Caspase-3酶活性为正常组的141%,与正常组比较,上述指标的差异均具有统计学意义。而N-乙酰半胱氨酸预处理后,细胞凋亡率降至26.0%,DHE和DHR123荧光强度分别为正常组的177%和290%,GSH含量为120 nmol/mg prot,线粒体膜电位为正常组的53%,Caspase-3酶活性为正常组的116%,与鱼藤酮组相比,差异均具有统计学意义。结论 N-乙酰半胱氨酸对PC12细胞具有保护作用,保护机制与其抗氧化活性有关。

Objective To investigate the potential protective effect of N-acetylcysteine（NAC）on rotenone-induced apoptosis of PC12 cells and the possible mechanisms.Methods PC12 cells were treated with 1 μmol/L of rotenone for 24 h to establish a cell model of Parkinson＇s disease（PD）.Based on this model,PC12 cells were pretreated with NAC（500 μmol/L）for 30 min before rotenone treatment.Apoptosis rate was analyzed by Annexin Ⅴ-FITC/PI flow cytometry（FCM）.Intracellular reactive oxygen species（ROS）was assessed by FCM using the fluorescent dyes,dihydroethidium（DHE）and dihydrorhodamine 123（DHR123）.The levels of reduced glutathione（GSH）were determined by using colorimetric method.Caspase-3 activity and mitochondrial membrane potential were also measured.Results After treatment with rotenone for 24 h,apoptosis rate of PC12 cells was reduced to 42.0%.The fluorescence intensity of DHE and DHR123 was increased to 230% and 333% of controls,respectively.GSH levels were reduced to 101 nmol/mg protein after rotenone treatment.Mitochondrial membrane potential was decreased to 46% of controls,and Caspase-3 activity increased to 141% of controls.The differences were all statistically significant as compared with control group.However,after pretreatment with NAC for 30 min,apoptosis rate of PC12 cells was 26.0%,and fluorescence intensity of DHE and DHR123 was 177% and 290% of controls,respectively.GSH levels were 120 nmol/mg protein.Mitochondrial membrane potential was 53% of controls,and Caspase-3 activity was 116% of controls.The differences were all statistically significant as compared with rotenone group.Conclusion These results demonstrated the protective action of NAC on PC12 cells in the rotenone-induced cell model of PD,possibly by alleviating the cell damage from oxidative stress with its antioxidant property.