Abstract

IMI causes inflammation and pyroptosis leading to pathological damage. • IMI targeting AMPK via SIRT3/PINK1 inhibited mitophagy and lysosomal function. • QUE alleviates IMI-induced mitophagy reduction, lysosomal dysfunction. Imidacloprid (IMI) is a pesticide widespreadly used due to its high efficiency, widely spectrum and usability. However, its high solubility contributes to significant environmental contamination impact on the health of non-target organisms, especially aquatic organisms. Current toxicological studies of IMI in aquatic organisms primarily focus on neurotoxicity, developmental toxicity, and bioaccumulation with limited research on its hepatotoxic mechanisms. Quercetin (QUE), a flavonoid known for its antioxidant properties, has garnered significant attention for its various beneficial biological effects. This study aims to elucidate the mechanism of IMI-induced damage to grass carp liver and alleviating effects of QUE. Using an in vivo model treated with 140 ng/L IMI and an in vitro model treated with 20 μmol/L IMI and/or 10 μmol/L QUE, determined that IMI exposure triggered oxidative stress, mitochondrion and lysosomal function decreased, and pyroptosis. Network Toxicology and Cellular Thermal Shift (CETSA) predicted and validated IMI bind 5′ adenosine monophosphate-activated protein kinase (AMPK) to interfere with its phosphorylation which indicates that AMPK is the target of IMI. Subsequently investigation revealed that QUE alleviates oxidative stress in grass carp hepatocytes induced by IMI by restoring mitochondrial and lysosomal function, recovering mitophagy flux, and mitigating pyroptosis, thereby restoring AMPK phosphorylation levels. This study revealed that IMI-induced grass carp hepatocyte damage causes pyroptosis via mitophagy flux blockage and uncovered the targets and mechanisms underlying QUE's mitigation of IMI toxicity. These results may offer insights into the development of therapeutic strategies to mitigate the harmful effect