α-Amanitin aggravates hepatic injury by activating oxidative stress and mitophagy via peroxiredoxin 6 inhibition

Abstract

Mushroom poisoning is mainly caused by α-amanitin (α-AMA), and there is currently no effective drug to treat α-AMA poisoning. Therefore, it is particularly important to find early diagnostic markers for α-AMA injury. Hepatic injury models induced by α-AMA were established both in hepatic cells and mice. The cell viability of human normal hepatic cells after α-AMA treatment was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Liver function parameters was assessed by the Enzyme-Linked Immunosorbent Assay (ELISA). Furthermore, oxidative stress was detected by 2',7'-Dichlorofluorescin Diacetate (DCFH-DA) and Dihydroethidium (DHE) staining. Autophagy- and apoptosis-related proteins were assessed by Western blot and immunofluorescence staining. We applied Hematoxylin and Eosin (H&E), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Oil Red O (ORO) staining to observe the degree of cell damage and hepatocyte apoptosis. In addition, mitochondrial membrane potential was also determined by JC-1 immunofluorescence staining and flow cytometry. The results showed that α-AMA decreased cell viability in a dose-dependent manner. In addition, the levels of alanine aminotransferase (ALT), aspartate transaminase (AST) and mitochondrial reactive oxygen species (mtROS) were observed to increase in the α-AMA-treated groups, whereas antioxidants superoxide dismutase (SOD) levels were reduced. Moreover, α-AMA promoted hepatocyte mitophagy and apoptosis, which were alleviated by PRDX6 overexpression. Finally, PRDX6 and Parkin were found to accumulate in mitochondria and α-AMA activated mitophagy by silencing PRDX6. Collectively, our results demonstrated that α-AMA activates oxidative stress and mitophagy by inhibiting the expression of PRDX6, leading to hepatic injury. These findings from both in vitro and in vivo models provide insights into the toxicological mechanisms of α-AMA, underscoring the potential of PRDX6 as a therapeutic target for treating α-AMA-induced hepatotoxicity. HIGHLIGHTS: α-AMA leads to ROS accumulation and activates oxidative stress. α-AMA promotes hepatocyte mitophagy and apoptosis. PRDX6 alleviates α-AMA-induced hepatic injury. PRDX6 mediates mitophagy through Parkin.

Keywords: Hepatic injury; PRDX6; Parkin; ROS; α-Amanitin.