GenX induces cochlear hair cell injury via System Xc^--Gpx4-Fsp1 ferroptosis axis

To investigate the role and mechanism of ferroptosis in cochlear hair cell injury induced by exposure to ammonium 2, 3, 3, 3-tetrafluoro-2-(heptafluoropropoxy)propanoate (GenX).

Mouse cochlear hair cell line HEI-OC1 was assigned to control, GenX, ferrostatin-1 (Fer-1), and GenX+Fer-1 groups. CCK-8 assay was used to assess the cytotoxicity of GenX and the rescue effect of Fer-1 co-treatment. Western blotting and qRT-PCR were employed to measure the protein and transcriptional expression of ferroptosis markers, cochlear function indicators, blood labyrinth barrier markers, and ferroptosis-related pathway. FerroOrange, Bodipy (C11), mitochondrial membrane potential assay kit (JC-1), and adenosine triphosphate (ATP) assay were applied to detect Fe²⁺ accumulation, lipid peroxidation, mitochondrial membrane dysfunction, and cellular ATP levels, respectively.

Exposure to 200 μmol/L GenX for 12 h significantly reduced the viability of HEI-OC1 cells (P<0.01), down-regulated the protein levels of glutathione peroxidase 4 (Gpx4), solute carrier family 7 member 11 (Slc7a11), and ferroptosis suppressor protein 1 (Fsp1) (P<0.05), whereas up-regulated acyl-coa synthetase long-chain family member 4 (Acsl4) (P<0.01), and decreased the expression of cochlear hair-cell function genes and blood labyrinth barrier genes (P<0.05). These changes were accompanied with Fe²⁺ accumulation, elevated lipid peroxidation, mitochondrial membrane damage, and reduced ATP production (P<0.001). Addition of Fer-1 restored cell viability (P<0.05), restored the expression of ferroptosis related proteins (P<0.05), and improved the expression of several hair-cell function and blood labyrinth barrier genes (P<0.05). In parallel, the GenX+Fer-1 group exhibited reduced Fe²⁺ accumulation, lower lipid peroxidation, attenuated mitochondrial membrane damage, and increased ATP level (P<0.001).

GenX induces iron-metabolism dysregulation and lipid peroxidation, and then leads to differentiation impairment of cochlear hair cells and barrier functions via the ferroptotic System Xc^--Gpx4-Fsp1 axis.