Abstract

Acetaminophen (APAP) intoxication is a common cause of liver injury. Silibinin has demonstrated potent hepatoprotective properties. However, its underlying mechanisms in APAP-induced liver injury (AILI) remain unclear. Autophagy is a critical adaptive response in AILI, contributing to the clearance of damaged mitochondria and the attenuation of oxidative stress. Therefore, we focused primarily on investigating the role of autophagy in mediating the hepatoprotective effects of silibinin. The effects of silibinin were evaluated in both AML12 cells and a C57BL/6J mouse model of AILI. Both the in vitro and in vivo experiments comprised four groups: a control group, an AILI model group, a silibinin treatment group, and a silibinin plus autophagy inhibitor group using PINK1-siRNA in cell culture and 3-Methyladenine in the animal experiment. Following induction of the AILI model in mice with APAP at a dose of 300 mg/kg, the animals received the designated interventions for five consecutive days. Histopathological alterations were assessed using hematoxylin–eosin staining. Hepatocyte proliferation and apoptosis were evaluated using the CCK-8 assay and immunohistochemical staining for Ki-67 and cleaved caspase-3, respectively, as well as ELISA for Cyclin D1. Liver function was assessed by serum biochemical analysis of alanine aminotransferase, aspartate aminotransferase, total bilirubin, and albumin. Mitochondrial oxidative stress–related parameters, including superoxide dismutase and malondialdehyde, were measured using colorimetric assays. The expression of autophagy-related genes and proteins (PINK1, Parkin, AMPK, LC3 and p62) was analyzed by quantitative PCR, immunofluorescence, and Western blotting. Transmission electron microscopy was employed to examine mitochondrial ultrastructure and the formation of autolysosomes in mouse liver tissue. In AML12 cells, silibinin mitigated AILI by activating the PINK1/Parkin pathway, thereby promoting mitophagy and enhancing cell pr