Oroxylin A ameliorates non-alcoholic fatty liver disease by modulating oxidative stress and ferroptosis through the Nrf2 pathway.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent and progressive liver disorder posing a global health challenge. Oroxylin A, a naturally occurring flavonoid, with a broad spectrum of pharmacological activities. This study aimed to explore the therapeutic potential of oroxylin A and unravel its molecular mechanisms in mitigating high-fat diet (HFD)-induced NAFLD in murine models. Wild-type (WT) and nuclear factor erythroid 2-related factor 2 knockout (Nrf2^-/-) mice were administered a HFD to generate in vivo models, while free fatty acids-treated HepG2 cells served as the in vitro model. To investigate the effects of oroxylin A, serum and liver biochemical markers, hepatic histology, lipid metabolism, and oxidative stress were assessed in a NAFLD mouse model. The underlying mechanisms of oroxylin A were further explored through Western blotting, immunohistochemistry, and immunofluorescence analysis. Oroxylin A mitigated hepatic steatosis and injury by reducing liver index, AST, ALT, TG, and TC levels, improving histology, and restoring lipid metabolism. Glucose and insulin tolerance tests demonstrated improved glucose homeostasis and insulin sensitivity. Moreover, oroxylin A suppressed inflammation, apoptosis, and fibrosis, while enhancing antioxidant defenses, and improving mitochondrial function. Mechanistically, oroxylin A activated the Keap1/Nrf2/GPX4/SLC7A11 axis, upregulating Nrf2 and HO-1. These effects were abolished in Nrf2^-/- mice. In vitro results were consistent, and molecular docking, dynamics simulations, and CETSA confirmed its direct Keap1 binding. Oroxylin A protects against NAFLD by modulating the Nrf2 pathway, reducing oxidative stress and ferroptosis, making it a promising candidate for clinical NAFLD therapy.