Metabolic dysfunction-associated steatohepatitis (MASH) is a severe, progressive liver disease lacking effective therapies. Disulfiram (DSF), an FDA-approved medication for alcohol dependence, exhibits diverse biological activities beyond its primary indication. This study aimed to evaluate whether DSF holds intervention promise for MASH and to unravel the underlying molecular mechanism. The efficacy of DSF was assessed in a mouse model of MASH induced by a choline-deficient, L-amino acid-defined diet, as well as in hepatocytes exposed to free fatty acids (FFAs) to trigger lipotoxicity. RNA-seq analysis combined with bioinformatic approaches was performed to identify key pathways and hub genes. Mechanistic validation was carried out using Western blotting and qPCR. Computational predictions suggested that DSF may influence insulin resistance, inflammation, autophagy-related markers, and lipid metabolism. In FFAs-treated hepatocytes, DSF administration dose-dependently reduced lipid accumulation and lipotoxicity. Consistently, in MASH mice, DSF administration significantly lowered elevated serum ALT (35%) and AST (40%) levels and the absolute hepatic triglyceride content (reduced from 1 to 0.5 μg/mg protein), and markedly attenuated hepatic steatosis, inflammation, fibrosis, and oxidative stress. Of note, RNA-seq analysis revealed that DSF modulated autophagy-related pathways and identified Aurora kinase A (AURKA) as a central downregulated hub gene. Mechanistically, DSF suppressed AURKA expression, which in turn led to changes in autophagy-related markers. These changes in autophagy-related markers were functionally coupled to a reduction in lipotoxicity. Collectively, DSF alleviates MASH by inhibiting AURKA, thereby relieving AURKA-mediated suppression of autophagy-related markers, which was associated with diminishing lipotoxicity, and ultimately achieving broad suppression of disease progression. Thus, DSF represents a promising hepatoprotective candidate for the intervention of MASH.
