N-乙酰半胱氨酸拮抗鱼藤酮致PC12细胞凋亡的研究

摘要

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(1 μmol/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(500 μmol/L)预处理PC12细胞30 min.采用Annexin Ⅴ-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 1 μmol/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 y-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 IlHR123 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 l4l％ 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 120nmol/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 rotenoneinduced cell model of PD, possibly by alleviating the cel! damage from oxidative stress with its antioxidant property.