Atractylenolide Ⅲ protects granulosa cells from oxidative injury by regulating the NOX2/NRF2 axis in polycystic ovary syndrome

NADPH oxidase 2 (NOX2) serves as a predominant source of reactive oxygen species (ROS), and excessive ROS accumulation leads to oxidative stress. Oxidative stress is a major factor in ovarian dysfunction in polycystic ovary syndrome (PCOS). Atractylenolide Ⅲ (ATⅢ) is a natural compound with antioxidant activity. The primary aim of this study was to investigate the role of NOX2 in ovarian dysfunction associated with PCOS and to evaluate the protective effects of ATⅢ. Primary ovarian granulosa cells (GCs) were harvested from patients with PCOS. The expression of apoptosis marker proteins, NOX2, and nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant-related genes and proteins were detected. A dihydrotestosterone (DHT)-induced KGN cell model was established to measure changes in oxidative stress after ATⅢ treatment. The interaction between ATⅢ and NOX2 was detected by molecular docking and cellular thermal shift assay (CETSA). GCs from PCOS patients showed increased apoptosis, increased NOX2 expression, and impaired antioxidant defense. DHT induction led to increased apoptosis, elevated ROS levels and NOX2 expression, and decreased NRF2 activity in KGN cells. ATⅢ treatment reversed these changes. ATⅢ also facilitates NRF2 nuclear translocation thereby elevating the expression of downstream heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) levels. Molecular docking and CETSA experiments supported the potential interaction and structural engagement between ATⅢ and NOX2. ATⅢ reversed the alterations in the NOX2 and NRF2 signaling pathways induced by CYBB (encoding NOX2) overexpression. These results indicate that ATⅢ reduces oxidative stress through modulation of the NOX2/NRF2 axis and restoring redox balance in GCs in PCOS.