Formononetin mitigates alcoholic liver disease by restoring ALDH2 function and inducing ERK1/2–Nrf2 antioxidant signaling

BACKGROUND: Alcohol consumption leads to the accumulation of acetaldehyde and reactive oxygen species (ROS), collectively contribute to the development of alcoholic liver disease (ALD). Formononetin (FN), a major isoflavone derived from Astragalus membranaceus, has demonstrated therapeutic potential in metabolic disorders. However, its role in ALD remains unknown. PURPOSE: This study aimed to investigate the hepatoprotective effect of FN in ALD and elucidate its underlying molecular mechanism. MATERIALS AND METHODS: ALD was induced in mice via chronic alcohol administration, with or without FN treatment. Liver histopathology, oxidative stress markers, acetaldehyde accumulation, mitochondrial function, and lipid profiles were assessed. Untargeted metabolomics and RNA sequencing were performed to identify FN-regulated pathways. Direct protein targets were identified using limited proteolysis-coupled mass spectrometry (LiP-MS) and virtual screening, validated by surface plasmon resonance (SPR) and microscale thermophoresis (CETSA). Functional validation was conducted using siRNA knockdown and pharmacological inhibitors. RESULTS: FN significantly attenuated alcohol-induced liver injury, fibrosis, lipid accumulation, and oxidative stress. It reduced hepatic ROS and acetaldehyde levels, improved mitochondrial integrity, and restored glutathione homeostasis. Metabolomics and transcriptomic analyses revealed that FN regulated NAD+ metabolism, cysteine and methionine metabolism, and activated the Nrf2 antioxidant pathway. Mechanistically, FN directly bound to aldehyde dehydrogenase 2 (ALDH2), enhancing its detoxifying activity. ALDH2 activation further promoted ERK1/2 and AP-1 phosphorylation, contributing to Nrf2-mediated glutathione biosynthesis and antioxidant defense. CONCLUSION: FN ameliorated alcohol-induced liver damage by directly targeting ALDH2 to promote acetaldehyde detoxification and indirectly activating the ALDH2-ERK1/2-Nrf2 axis to enhance antioxidant capacity. FN might serve as a promising therapeutic candidate for the treatment of ALD.