Autophagy Promotes α-Amanitin-Induced Apoptosis of Hepa1-6 Liver Cells
Autophagy Promotes α-Amanitin-Induced Apoptosis of Hepa1-6 Liver Cells
复制标题
自噬促进 α-鹅膏菌素诱导的 Hepa1-6 肝细胞凋亡。
DOI：
10.1021/acs.chemrestox.1c00297
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发表时间：
2022-03-21
影响因子：
4.1
通讯作者：
Qu, Weijie
中科院分区：
文献类型：
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作者：
Gu, Xiaolong;Zhang, Limei;Qu, Weijie
据估计，中国90%的食物中毒死亡可归因于鹅膏菌中毒，其主要毒素是α-鹅膏毒肽。研究表明，细胞凋亡在α-鹅膏蕈碱诱导的肝损伤中起关键作用。虽然在不同的肝脏模型中自噬和细胞凋亡之间的关系已经被解决了很多次，但自噬对α-鹅膏蕈碱诱导的细胞凋亡是否起着有利或不利的作用尚未阐明。因此，本研究旨在探讨自噬在α-鹅膏蕈碱诱导Hepa 1 -6肝细胞凋亡中的作用。采用MTT法检测细胞活力，2 ′，7 ′-二氯荧光素二乙酸酯探针检测活性氧（ROS）水平，流式细胞仪检测细胞凋亡，丹磺尸胺（MDC）染色观察细胞自噬，Western印迹法检测细胞凋亡和自噬蛋白。结果表明，α-鹅膏蕈碱以时间和浓度依赖性方式抑制细胞活力。此外，ROS的释放随着α-鹅膏蕈碱量的增加而增加。观察到细胞凋亡和自噬，其特征在于在荧光显微镜下凋亡率和自噬囊泡增加，以及Bax/Bcl-2、裂解的半胱天冬酶-3和LC 3-II/I的上调和p62的下调。自噬激活剂雷帕霉素（rapamycin，Rap）和自噬抑制剂3-甲基胺（3-methylademine，3-MA）的加入，可显著提高细胞凋亡率、Bax/Bcl-2比值和caspase-3蛋白表达水平，而3-MA则显著降低。此外，发现细胞活力随着自噬的促进而进一步降低。值得注意的是，自噬升高后ROS水平减弱。结论：自噬可促进α-鹅膏蕈碱诱导的Hepa 1 -6细胞凋亡，且该过程与ROS水平无关。本研究为α-鹅膏蕈碱致肝损伤的毒理学机制研究提供了理论依据。
It is estimated that 90% of deaths from food poisoning in China can be attributed to Amanita poisoning, whose main toxin is alpha-amanitin. Studies showed that apoptosis plays a critical role in liver injuries induced by alpha-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 alpha-amanitin-induced apoptosis has not been clarified. Therefore, this study was conducted to explore the effect of autophagy in alpha-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 alpha-amanitin-induced apoptosis and autophagy, respectively, and apoptosis and autophagy proteins were assessed by western blotting. The results showed that alpha-amanitin suppressed cell viability in a time- and concentration-dependent manner. Moreover, the release of ROS was increased with increasing alpha-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 alpha-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 alpha-amanitin-induced liver injuries.