The Study On Alcohol-induced Autophagy In PC12 Cells

Objective The study is to investigate the molecular mechanisms and the relationship between autophagy and apoptosis that alcohol induces in rat pheochromocytoma cell line (PC12), in order to provide data support for the research of alcohol-induced neurotoxicity.Methods The growing PC12 cells were divided into 5 groups: normal control group, serum-deprived group, 50mmol·L-1 alcohol-induced group, 100mmol·L-1 alcohol-induced group and 200mmol·L-1 alcohol-induced group. In the research, cell model is established by adding different dose alcohol into its medium to damage the PC12 cells. The proliferation of PC12 cells and the inhibiting effect of alcohol on PC12 cell proliferation were examined by MTT assay. Immunofluorescence technique was performed to observed apoptotic cell morphology with Hoechst33258 labeling. The expression of microtubule-associated protein 1 light chain 3 (LC3) in PC12 cells were observed by immunofluorescence staining. Autophagic and apoptotic ratio of PC12 cells were determined by HCS (High Content Screening). Western blot analysis was used to assay the expression of apoptosis and autophagy-related proteins. All experimental values are expressed as means±SD. To determine statistical significance, the values were compared using one-way analysis of variance (ANOVA), two-group t-tests with differences considered significant for P values less than 0.05.Results The results of MTT assay indicated that alcohol possessed inhibiting activity to proliferation of PC12 cells. Alcohol inhibited the proliferation of PC12 cells within the range of 100mmol·L-1~400mmol·L-1. Compared with the controls, more cells appeared such cell shrinkage, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies as classic apoptotic features. More autophagosomes in PC12 cells were observed by immunofluorescence staining in dose-dependent manner and negative time-dependent manner. Moreover a large number quantity of autophagosomes was observed in alcohol-induced PC12 cells. The cell ultrastructure was examined 2h after alcohol-induced PC12 cell by transmission electron microscopy. The results of HCS assay revealed that apoptotic ratio was in dose- and time-dependent manners. Autophagic ratio was also in dose- and time-dependent manners. Compared with the normal control group, expression of LC3, Beclin-1, Bcl-2 and P53 in PC12 cells induced by different dose alcohol increased significantly, and in dose-dependent manner.Conclusions1.Alcohol possessed inhibiting activity to proliferation of PC12 cells. The inhibiting effect was significant within the range of 100mmol·L-1~400mmol·L-1.2.Alcohol at 50 mmol·L-1~200mmol·L-1possessed inhibiting activity to apoptosis of PC12 cells.3.Alcohol possessed inhibiting activity to autophagy of PC12 cells, and the autophagy ratio was highest in 2h.4.The autophagy and apoptosis in alcohol-induced PC12 cells had high correlation.