Liu Liping, Sun Tiedong, Xin Feng, Cui Wei, Guo Jianquan, Hu Jian
The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin, Heilongjiang 150001, PR China.
School of Life Science and Technology, Harbin Institute of Technology, 2 Yikuang Street, Nangang District, Harbin, Heilongjiang 150080, PR China.
Alcohol Alcohol. 2017 Jan;52(1):12-18. doi: 10.1093/alcalc/agw077. Epub 2016 Oct 19.
To study whether autophagy participates in the neuroprotective effect of nerve growth factor (NGF) on neurons treated with alcohol.
The autophagy-related markers were used to explore the role of autophagy in PC12 cells exposed to alcohol or pre-incubated with NGF before initiating the treatment with alcohol (100 mM; 6 h). PC12 cells were pre-incubated with 3-methyladenine (3-MA) (10 mM; 1 h) or rapamycin (100 nM; 1 h) before co-incubated with alcohol (100 mM; 6 h) in order to investigate the relationship between apoptosis and autophagy. PC12 cells were pre-incubated with LY294002 (50 μM; 30 min) before co-incubated with NGF and alcohol in order to analyze the protein expression of PI3K/Akt/mTOR pathway via western blotting.
By methylthiazoltetrazolium, western blotting and flow cytometry assays, we found that cell viability decreased in a dose- and time-dependent manner after treatment with alcohol in PC12 cells. As cells were exposed to alcohol, the levels of LC3-II proteins became elevated, likewise, pre-treatment with 3-methyladenine (3-MA, an autophagic inhibitor) or rapamycin (an autophagic inducer) resulted in an increased or decreased percentage of apoptosis in contrast to other alcohol-treated groups, respectively. NGF markedly increased LC3-II production after treatment with alcohol in a dose-dependent manner. Moreover, NGF remarkably attenuated the phosphorylation effect of alcohol exposure on PI3K/Akt/mTOR pathway, which was suppressed by LY294002 (Akt inhibitor).
NGF protects against alcohol-induced neurotoxicity via PI3K/Akt/mTOR pathway.
In this study, we chose the PC12 cell line as a neuronal model, and our results demonstrate that nerve growth factor can induce autophagy with the neuroprotective effect and regulatory mechanisms of alcohol-induced autophagy in PC12 cells.
研究自噬是否参与神经生长因子（NGF）对酒精处理的神经元的神经保护作用。
使用自噬相关标志物来探讨自噬在暴露于酒精或在用酒精（100 mM；6小时）处理前用NGF预孵育的PC12细胞中的作用。为了研究凋亡与自噬之间的关系，PC12细胞在用酒精（100 mM；6小时）共孵育前先用3-甲基腺嘌呤（3-MA）（10 mM；1小时）或雷帕霉素（100 nM；1小时）预孵育。为了通过蛋白质印迹分析PI3K/Akt/mTOR途径的蛋白质表达，PC12细胞在用NGF和酒精共孵育前先用LY294002（50 μM；30分钟）预孵育。
通过甲基噻唑基四氮唑、蛋白质印迹和流式细胞术分析，我们发现PC12细胞在用酒精处理后细胞活力呈剂量和时间依赖性下降。随着细胞暴露于酒精，LC3-II蛋白水平升高，同样，与其他酒精处理组相比，用3-甲基腺嘌呤（3-MA，一种自噬抑制剂）或雷帕霉素（一种自噬诱导剂）预处理分别导致凋亡百分比增加或减少。在用酒精处理后，NGF以剂量依赖性方式显著增加LC3-II的产生。此外，NGF显著减弱了酒精暴露对PI3K/Akt/mTOR途径的磷酸化作用，而LY294002（Akt抑制剂）抑制了该作用。
NGF通过PI3K/Akt/mTOR途径保护细胞免受酒精诱导的神经毒性。
在本研究中，我们选择PC12细胞系作为神经元模型，我们的结果表明神经生长因子可以诱导自噬，并具有神经保护作用以及对PC12细胞中酒精诱导的自噬的调节机制。
