H_2O_2预处理对多巴胺损伤PC12细胞的适应性保护作用

Adaptive protection of H_2O_2 preconditioning against dopamine-induced damage in PC12 cells

目的探讨H2O2预处理对多巴胺(dopamine,DA)损伤PC12细胞的适应性保护作用。方法透射电镜、Hoechst染色和PI染色流式细胞仪(flowcytometry,FCM)观测细胞凋亡,MTT代谢率法观察细胞线粒体氧化磷酸化功能状态,Rh123染色FCM检测细胞线粒体膜电位(△Ψm)。结果50μmol·L-1DA作用PC12细胞24h后,电镜可观察到PC12细胞体积缩小,核染色质浓缩、边集,核碎裂等细胞凋亡征象。Hoechst33258染色显示,DA(50μmol·L-1)作用24h后,经H2O2预处理的PC12细胞的凋亡量较未预处理的明显减少。50、100和200μmol·L-1DA作用24h后,PC12细胞的凋亡率分别为(20.9±1.8)%、(40.5±6.4)%、(88.1±3.9)%,而经H2O2预处理的PC12细胞的凋亡率分别下降至(4.9±2.9)%、(12.0±1.4)%、(61.5±3.4)%(P<0.01)。20、40、80μmol·L-1DA作用细胞24h后,细胞MTT代谢率明显下降,而H2O2预处理抑制20、40、80μmol·L-1DA引起的PC12细胞MTT代谢率的下降(P<0.01)。50μmol·L-1DA作用24h后,PC12细胞的平均Rh123荧光强度由正常对照的(46.87±0.33)下降至(4.39±2.93),而经H2O2预处理的PC12细胞,其平均Rh123荧光强度仅下降到(10.50±0.28),下降程度明显减轻(P<0.01)。结论H2O2预处理对DA损伤PC12细胞具有适应性保护作用。

Aim To study the adaptive protection of H_2O_2 preconditioning against dopamine-induced damage in PC12 cells.Methods The apoptosis of PC12 cells was observed by electron microscope, PI stain flow cytometry (FCM) and Hoechst stain. The mitochondrial energy redox state was measured by MTT assay. The mitochondrial membrane potential(△Ψm) was investigated by the fluorescent probe of rhodamine 123.Results PC12 cells exposed to 50 μmol·L -1 DA showed chromatin condensation and nucleus fragmentation observed by electron microscope. Exposure to DA (50 μmol·L -1) for 24 h, the apoptosis of PC12 cells preconditioned by 10 μmol·L -1 H_2O_2 for 90 min as measured by Hoechst stain was significantly decreased,compared with no-preconditioned cells. Exposure to 50,100,and 200 μmol·L -1 H_2O_2 for 24 h, the proportion of apoptosis of PC12 cells was decreased from (20.9±1.8)%, (40.5±6.4)% and (88.1±3.9)% to (4.9±2.9)%, (12.0±1.4)%, (61.5±3.4)% after H_2O_2 preconditioning, respectively. By exposuring PC12 cells to 20,40,and 80 μmol·L -1 DA for 24 h, the rates of MTT metabolism were reduced and the effect was prevented by H_2O_2 preconditioning. After 50 μmol·L -1 DA exposure for 24 h,the mean fluorescence intensity of rhodamine 123 in no-preconditioned PC12 cells was decreased from (46.87±0.33) to (4.39±2.93),and that of preconditioned PC12 cells was decreased from (46.87±0.33) to (10.50±0.28). Conclusion H_2O_2 preconditioning possesses adaptive protection against dopamine-induced damage in PC12 cells.