Polycystic ovary syndrome (PCOS) is a complex endocrine disorder frequently associated with granulosa cell dysfunction. However, its underlying molecular mechanisms remain unclear. Therefore, we aimed to examine the role of APPL1 in PCOS and determine whether it attenuates oxidative stress and ferroptosis by modulating mitophagy in granulosa cells. An in vitro PCOS model was established by treating KGN cells with dehydroepiandrosterone (DHEA), with or without concurrent APPL1 overexpression. An in vivo PCOS rat model was established by DHEA injection, followed by intraovarian lentiviral delivery to overexpress APPL1. Oxidative stress, ferroptosis, and mitophagy markers were also evaluated. Mdivi-1, the DRP1/mitochondrial fission inhibitor, was used to examine this pathway. APPL1 expression was significantly reduced in DHEA-treated KGN cells and PCOS rat ovaries. APPL1 overexpression markedly attenuated DHEA-induced oxidative stress and ferroptosis in vitro, as evidenced by reduced ROS, MDA, 8‑OHdG, Fe²⁺, and ACSL4 levels, together with restored antioxidant enzyme activities and GPX4/SLC7A11 expression. Concurrently, APPL1 overexpression enhanced mitophagy, as shown by an increased LC3-II/LC3-I ratio and decreased p62 and TOMM20/COX IV levels. Treatment with Mdivi-1 attenuated these protective effects. APPL1 overexpression improved estrous cyclicity, attenuated hyperandrogenism, ameliorated ovarian morphology, and alleviated ovarian oxidative stress and ferroptosis in PCOS rats. APPL1 protected granulosa cells against DHEA-induced oxidative injury and ferroptosis by enhancing mitophagy in preclinical PCOS models. These findings provide mechanistic insights into PCOS pathogenesis and identify the APPL1-mitophagy axis as a potential target for PCOS intervention.
