FMO3 exacerbates hepatic endoplasmic reticulum stress in drug-induced liver injury by inhibiting CREB3/P4HB axis and activating TMAO-mediated PERK pathway.

Objective: The primary objective of this study is to elucidate the role of FMO3, an important enzyme in drug metabolism, and its metabolites in Drug-induced liver injury (DILI).

Methods: We overexpressed hepatic FMO3 in mice by injecting AAV8 to examine their liver morphology under acetaminophen (APAP) or monocrotaline (MCT) treatment. We also detected the metabolite TMAO of FMO3 in patients and mice with DILI, and further verified its regulatory effects on the endoplasmic reticulum stress pathway in hepatocytes through in vivo and in vitro experiments.

Results: We found that FMO3 is upregulated in patients and male mice with DILI and overexpression of hepatic FMO3 exacerbates APAP or MCT-induced acute liver injury in mice. Mechanistically, FMO3 binds to endoplasmic reticulum (ER) stress-related transcription factor CREB3 (cAMP response element-binding protein 3) and inhibits its nuclear transcription. The decreased activity of CREB3 reduces the expression of the downstream gene P4HB(prolyl 4-hydroxylase subunit beta), subsequently inducing ER stress and apoptosis. Trimethylamine N-Oxide (TMAO), as a metabolite of FMO3, is also significantly elevated in patients with pyrrolizidine alkaloids-induced acute liver injury and APAP or MCT-induced liver injury in male mice. TMAO triggers ER stress by activating the PERK signaling pathway, and inhibiting TMAO production in DILI mice mitigates liver injury.

Conclusions: Overall, the above findings identify FMO3 as a potential enzyme that facilitates the progression of DILI and exerts ER stress by CREB3/P4HB axis and its metabolites TMAO, which presents new therapeutic targets for DILI.