Reverse relationship between autophagy and apoptosis in an in vitro model of cortical neuronal injury.

Autophagy and apoptosis are intertwined, and their relationship involves complex cross-talk. Whether the activation and inhibition of autophagy protect or damage neurons in the central nervous system has been a matter of longstanding controversy. We investigated the effect of autophagy on the apoptosis of cortical neurons after oxygen- and glucose-deprivation/reoxygenation (OGD/R) injury in vitro and found that protective mechanism activation was the predominant response to enhanced autophagy activation and increased autophagic flux. After successful establishment of an OGD/R model with cortical neurons, the autophagy activator rapamycin (Rap) or the late-autophagy inhibitor bafilomycin A1 (BafA1) was added to cell groups according to the experimental design. Cell viability was determined by Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays, and the apoptosis rate was measured by analysing Annexin V-FITC/PI-stained cells. The protein and mRNA expression levels of the apoptosis factors Caspase8 and Caspase3 and autophagy-associated proteins LC3 and p62 were measured by Western blotting and RT-qPCR. The extent of autophagic flux was determined by measuring the intensity of double immunofluorescence labelled protein after cells were transfected with RFP-GFP-LC3-expressing virus, and the ultrastructures of autophagosomes were observed by transmission electron microscopy (TEM). The results showed that cell viability decreased and that cells underwent autophagy and apoptosis after OGD/R. After the addition of Rap, cell viability was increased, and the apoptosis rate was decreased significantly. In addition, the level of the autophagic flux protein LC3II was increased, and the level of p62 was decreased. The number of autophagosomes and the ratio of autophagosomes to lysosomes were increased significantly. After BafA1 intervention, however, these results were reversed, with decreased cell viability, a significantly increased apoptosis rate, and disrupted autophagic flux. In conclusion, enhanced autophagy activation or autophagic flux exerted a significant protective effect on neurons after OGD/R injury in vitro.