Mechanisms and interventions of Aeromonas hydrophila-induced hepatic injury in Megalobrama amblycephala: Focus on oxidative stress-mediated mitochondrial apoptosis

Abstract

Our previous study demonstrated that Aeromonas hydrophila induces apoptosis and tissue injury in Megalobrama amblycephala. In this study, we explored the specific apoptotic pathways activated by A. hydrophila. Results revealed significant A. hydrophila enrichment in hepatocytes post-infection, which was attenuated by lipid raft inhibitors (methyl-β-cyclodextrin and simvastatin), along with cholesterol depletion. Meanwhile, a significant accumulation of total reactive oxygen species (T-ROS) in hepatocytes was observed, and the increase in T-ROS positive cells was further confirmed by flow cytometry. Additionally, A. hydrophila infection reduced mitochondrial membrane potential (MMP) and increased hepatocyte apoptosis. Western blot analysis revealed decreased mitochondrial cytochrome c and increased cytoplasmic cytochrome c levels after infection, along with significant activation of caspase-9 and caspase-3. To determine whether oxidative stress mediates A. hydrophila induced mitochondrial apoptosis, hepatocytes were pretreated with N-acetylcysteine (NAC) before infection. NAC treatment significantly reduced T-ROS accumulation, alleviated MMP decline and cytochrome c release, and inhibited caspase-induced apoptosis. In vivo experiment similarly demonstrated that NAC significantly attenuated A. hydrophila-induced mitochondrial damage, apoptosis pathway activation, and hepatic tissue vacuolization. In conclusion, this research enhances our understanding of A. hydrophila's pathogenic mechanism and provides a foundation for developing strategies to prevent and treat infections caused by this bacterium in fish.