Autophagy Promotes α-Amanitin-Induced Apoptosis of Hepa1-6 Liver Cells.

ABSTRACT

It is estimated that 90% of deaths from food poisoning in China can be attributed to Amanita poisoning, whose main toxin is α-amanitin. Studies showed that apoptosis plays a critical role in liver injuries induced by α-amanitin. Although the relationship between autophagy and apoptosis in different liver models has been addressed many times, whether autophagy plays a pro or con effect on α-amanitin-induced apoptosis has not been clarified. Therefore, this study was conducted to explore the effect of autophagy in α-amanitin-induced apoptosis in Hepa1-6 liver cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was applied to determine cell viability, a 2',7'-dichlorofluorescin diacetate probe was used to monitor reactive oxygen species (ROS) levels, a flow cytometer and dansylcadaverine (MDC) staining were used to observe α-amanitin-induced apoptosis and autophagy, respectively, and apoptosis and autophagy proteins were assessed by western blotting. The results showed that α-amanitin suppressed cell viability in a time- and concentration-dependent manner. Moreover, the release of ROS was increased with increasing α-amanitin amount. Cell apoptosis and autophagy were noticed and characterized by the increased apoptosis rate and autophagic vesicles under a fluorescence microscope as well as upregulation of Bax/Bcl-2, cleaved caspase-3, and LC3-II/I and downregulation of p62. Further, the autophagy activator rapamycin (Rap) and the inhibitor 3-methylademine (3-MA) were introduced, which showed that the apoptosis rate and the ratio of Bax/Bcl-2 as well as the protein expression level of cleaved caspase-3 increased significantly with the pretreatment of Rap and decreased remarkably with the pretreatment of 3-MA. Moreover, cell viability was found to decrease further with the promotion of autophagy. Notably, the ROS level was attenuated after autophagy was elevated. In conclusion, autophagy could promote α-amanitin-induced Hepa1-6 cell apoptosis, and the process is unassociated with ROS levels. This research provides a theoretical basis for the study of the toxicological mechanism of α-amanitin-induced liver injuries.