Biochanin A alleviates HFD-induced MAFLD by inhibiting IRE1α-SPT-ceramide axis and improving intestinal homeostasis.

Metabolic-associated fatty liver disease (MAFLD) progresses via a vicious cycle of "lipid dysregulation-ceramide-inflammation-oxidative stress-ferroptosis," with sodium palmitate (PA) as a key mediator of hepatic lipotoxicity. To screen ameliorative natural compounds, we performed high-throughput screening of 236 traditional Chinese medicine-derived compounds using PA-induced AML-12 hepatocytes, identifying biochanin A (BCA)-a major isoflavone in chickpeas-as a potent protector against hepatocyte death. We validated BCA's effects in vitro (PA-induced AML-12 cells) and in vivo (high-fat diet-induced MAFLD mice, 25/50 mg/kg BCA), combined with IRE1α agonist IXA4 rescue experiments and multi-omics analyses. A novel finding is that BCA directly binds IRE1α (via LEU23/CYS91, validated by molecular docking and 100-ns MD simulations) and specifically inhibits the IRE1α-SPT-ceramide axis. This downregulates SPTLC1/SPTLC2 (ceramide synthesis rate-limiting enzymes), normalizes hepatic C16/C24 ceramide levels, suppresses IL-1β/IL-6 production, restores mitochondrial OXPHOS function, reduces ROS/lipid peroxidation, and inhibits ferroptosis. Concurrently, BCA treatment was associated with alterations in gut microbiota composition (enriching Bacilli, reducing pro-inflammatory Coriobacteriia), enhanced intestinal barrier function, and reduced LPS translocation, which may contribute to mitigating hepatic inflammation. Notably, IXA4 completely reversed BCA's protective effects. In conclusion, BCA ameliorates MAFLD by inhibiting the IRE1α-SPT-ceramide axis to block the pathological cascade. In parallel, BCA treatment is associated with alterations in gut microbiota composition, improved intestinal barrier integrity, and reduced systemic inflammation, suggesting that the gut-liver axis may be involved in its protective effects.