ELANE enhances KEAP1 protein stability and reduces NRF2-mediated ferroptosis inhibition in metabolic dysfunction-associated fatty liver disease.

Neutrophil elastase (Elane) is upregulated in metabolic-associated fatty liver disease (MAFLD) and has the capacity to promote disease progression. However, the mechanism by which Elane promotes MAFLD development remains unclear. Ferroptosis, which is an iron-dependent nonapoptotic form of cell death characterized by the iron-induced accumulation of lipid reactive oxygen species (ROS), has been recently considered as an important mechanism for the development of MAFLD. In this study, we used mice of Elane-knockout (Elane-KO) and wild-type (WT), and their primary mouse hepatocytes to establish MAFLD models in vivo and vitro for elucidating the role of Elane in ferroptosis of hepatocytes and MAFLD development. Elane-KO in vivo reduced high-fat diet (HFD) induced hepatic lipid peroxidation levels and the proportion of hepatocyte death, upregulated the expression of Nrf2 and Gpx4, and downregulated Keap1 expression. Treatment with recombinant Elane increased the lipid peroxidation level of hepatocytes, increased the ferroptosis rate of hepatocytes, upregulated the expression of Keap1, enhanced the ubiquitination of Nrf2, and downregulated the expression of Nrf2 and Gpx4 in an FFA-induced MAFLD in vitro model. However, primary hepatocytes from Elane-KO mice presented opposite changes. Furthermore, an in vitro experiment revealed that Elane enhanced the protein stability of Keap1 and thus increased Keap1 expression in hepatocytes by inhibiting the lysosomal degradation of the Keap1 protein. Finally, in vitro Co-IP experiments revealed that Elane increased the protein stability of Keap1 by weakening the binding between P62 and Keap1 and ultimately promoted hepatocyte Nrf2 ubiquitination and ferroptosis in MAFLD. In conclusion, our results suggested that Elane promoted hepatocyte ferroptosis in MAFLD through the P62-Keap1-Nrf2-Gpx4 axis. Elane promotes ferroptosis in hepatocytes from fatty livers. Elane reduces the binding of P62 to Keap1, thereby increasing Keap1 protein stability and subsequently inhibiting the Nrf2/Gpx4 pathway, ultimately leading to ferroptosis in hepatocytes.