多索茶碱通过激活 AMPK 通路改善过氧化氢诱导的PC12细胞氧化损伤

Doxofylline attenuates hydrogen peroxide-induced oxidative damage in PC12 cells by activating AMPK pathway

关键词:  多索茶碱  过氧化氢  PC12细胞  氧化应激  AMPK信号通路

Key words:  doxofylline  hydrogen peroxide  PC12 cells  oxidative stress  AMPK signaling pathway

摘要:

目的 研究多索茶碱对过氧化氢 (H2O2) 诱导的PC12细胞毒性的保护作用及其潜在机制。方法 采用 H2O2 建立细胞氧化损伤模型。MTT检测细胞活力；Tunel染色、流式细胞仪、Caspase3活性试剂盒检测细胞凋亡；乳酸脱氢酶（LDH）试剂盒检测细胞膜损伤；线粒体膜电位检测试剂盒（JC-1）和DCFH-DA试剂检测线粒体功能变化；蛋白质免疫印迹用于检测信号通路蛋白水平的变化。结果 多索茶碱浓度依赖性改善了 H2O2诱导的PC12细胞内活性氧（ROS）的异常水平，恢复了线粒体膜电位，降低了Caspase-3激活，进而降低了PC12细胞凋亡。多索茶碱还以时间和浓度依赖性方式刺激 PC12 细胞中AMPK的磷酸化，其保护作用可被AMPK通路抑制剂逆转。结论 多索茶碱通过激活AMPK信号通路改善H2O2诱导的PC12细胞损伤。

Abstract:

OBJECTIVE To investigate the protective effect of doxofylline against hydrogen peroxide (H2O2)-induced cytotoxicity in PC12 and its underlying mechanism. METHODS The cell oxidative damage model was established by H2O2. MTT assay was used to detect the cell viability; Tunel staining, flow cytometry and caspase3 activation kit were used to detect cell apoptosis; lactate dehydrogenase kit to detect cell membrane damage; mitochondrial membrane potential detection kit (JC-1) and DCFH-DA reagent were used to detect changes in mitochondrial function; Western blotting was used to detect changes in signaling pathway protein levels. RESULTS Doxofylline concentration-dependently improved H2O2-induced abnormal levels of reactive oxygen species (ROS) in PC12 cells, restored mitochondrial membrane potential, reduced Caspase-3 activation, and finally reduced PC12 cell apoptosis. Doxofylline also stimulated phosphorylation of AMPK in PC12 cells in a time- and concentration-dependent manner, and its protective effect was reversed by AMPK pathway inhibitors. CONCLUSION Doxofylline improves H2O2-induced PC12 cell injury by activating AMPK signaling pathway.