FDX1-mediated cuproptosis promotes cholestatic liver injury exacerbated by taurocholic acid-enhanced copper accumulation.

Cholestatic liver injury, characterized by direct exposure of hepatocytes to retained bile components with elevated concentrations, represents a common manifestation of various hepatobiliary disorders with persistent threats to long-term patient survival despite existing therapies. As the primary route for copper elimination, cholestasis raises questions about the role of copper in cholestatic liver injury and its specific molecular mechanisms. Our single-center retrospective study revealed elevated serum copper levels in subjects with increased gamma-glutamyl transferase compared to controls. Single-cell sequencing of biliary atresia (BA) patients' cholestatic liver specimens demonstrated downregulation of FDX1, a key cuproptosis marker, in BA hepatocytes. Bile duct-ligated rats under high-copper diets exhibited accelerated liver injury, attenuated by copper chelator tetrathiomolybdate (TTM). In vitro, copper chloride/elesclomol-induced DLAT monomer reduction and oligomerization alongside impaired lipoylation. Given the special coexistence of copper overload and accumulated bile components within the hepatic microenvironment, notably, we found that taurocholic acid potentiated hepatic copper accumulation under cholestatic conditions. Mechanistically, transcriptomic analysis implicated smoothened signaling inhibition in cuproptosis progression, with smoothened agonist (SAG) restoring DLAT expression and cellular viability. Interestingly, FDX1 overexpression enhanced cuproptosis resistance of hepatocytes through DLAT monomer stabilization and LIAS-mediated lipoylation. Cholestasis-induced copper overload drives liver injury via taurocholic acid-exacerbated and FDX1-mediated cuproptosis. Our findings propose TTM and SAG as therapeutic candidates and reveal complex FDX1 regulatory roles, suggesting novel approach for managing cholestatic liver injury.