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

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、Caspase-3活性试剂盒检测细胞凋亡;乳酸脱氢酶试剂盒检测细胞膜损伤;JC-1染色检测线粒体膜电位和DCFH-DA试剂检测细胞内ROS的产生;Western blotting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。多索茶碱刺激PC12细胞中AMPK的磷酸化,抑制AMPK信号通路其保护作用可被逆转。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。

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 Caspase-3 activation kit were used to detect cell apoptosis;Lactate dehydrogenase kit was used to detect cell membrane damage;Mitochondrial membrane potential was detected by JC-1 staining and the intercellular ROS level was detected by DCFH-DA staining;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 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,and its protective effect was reversed by AMPK pathway inhibitors.CONCLUSION Doxofylline improves H2O2-induced PC12 cell injury by activating AMPK signaling pathway.