Hydroxynonenal causes hepatocyte death by lysosomal disintegrity in NAFLD.

Background: The mechanisms underlying hepatocyte death and the role of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in non-alcoholic fatty liver disease (NAFLD) remains unclear. Methods: Changes in cell morphology and intracellular organelles after HNE treatment were observed in hepatocellular carcinoma cell lines, using time-lapse imaging, electron microscopic observation, and immunofluorescent staining. Calpain activation and heat shock protein 70.1 (Hsp70.1) cleavage were analyzed by western blotting. These in vitro experiments were revalidated using liver tissue from HNE-treated Japanese macaque and 90 NAFLD patients. Results: Time-lapse imaging demonstrated that HNE induced “bursting” cell death with the gradual reduction and loss of lysosomes in hepatoma cell lines. Electron microscopic observation revealed that HNE disrupted the lysosomal membrane, and immunofluorescent staining showed the leakage of its content cathepsin B. These were associated with rupture/permeabilization of the lysosomal membrane resulting from activation of μ-calpain and cleavage of Hsp70.1. These results were confirmed in HNE-treated Japanese macaque and human NAFLD liver tissue. The degree of HNE deposition in human NAFLD hepatocytes was more severe in cases with high lobular inflammation, ballooning, and fibrosis scores. Furthermore, increased HNE deposition was associated with enlargement of the stained area of lysosomes in hepatocytes. Conclusions: HNE causes lysosomal rupture/permeabilization and contributes to cell death in NAFLD. These results provide novel insights into the mechanism of hepatocyte death and will contribute to the development of new therapeutic strategies for NAFLD.