Objective To examine the role of Cd-induced reactive oxygen species（ROS） generation in the apoptosis of neuronal cells. Methods Neuronal cells（primary rat cerebral cortical neurons and PC12 cells） were incubated with or without Cd post-pretreatment with rapamycin（Rap） or N-acetyl-L-cysteine（NAC）. Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B（Akt）/mammalian target of rapamycin（m TOR） and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase（p70 S6K）, and eukaryotic initiation factor 4E binding protein 1（4E-BP1）. Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein（Bcl-2/Bax） ratio, release of cytochrome c, cleavage of caspase-3 and poly（ADP-ribose） polymerase（PARP）, and nuclear translocation of apoptosis-inducing factor（AIF） and endonuclease G（Endo G）. Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.
YUAN YanWANG YiHU Fei FeiJIANG Chen YangZHANG Ya JingYANG Jin LongZHAO Shi WenGU Jian HongLIU Xue ZhongBIAN Jian ChunLIU Zong Ping
PC12细胞培养过程中添加镉和/或PI3K抑制剂LY294002,免疫印迹法检测Beclin-1复合物和LC3-II的表达变化,研究其在自噬中的作用。结果表明:20μmol·L-1镉处理不同时间后,Beclin-1、class III PI3K和Bcl-2的表达总体呈现先升高后降低的趋势;联合LY294002后显著抑制了Beclin-1,class III PI3K、Bcl-2和LC3-II的表达水平(P<0.05或P<0.01)。说明镉暴露PC12细胞后,促使Bcl-2/Beclin-1复合物解离,Beclin-1进一步和class III PI3K结合,参与调节自噬。
