Aspartame triggers premature ovarian failure via SIRT1-dependent suppression of the PI3K/AKT/mTOR pathway and induction of oxidative stress and apoptosis.

Given concerns about the reproductive safety of aspartame, this study investigated aspartame-induced premature ovarian failure (POF) using integrated computational biology, in vitro, and in vivo approaches. Network toxicology identified 182 overlapping targets, which were mainly enriched in oxidative stress (OS), apoptosis, and the PI3K/AKT/mTOR pathway. Molecular dynamics (MD) simulations initially predicted that SIRT1 had the highest binding affinity for aspartame, and this interaction was further validated by surface plasmon resonance (SPR) analysis. In vitro experiments using KGN cells showed that aspartame suppressed cell proliferation in a time- and concentration-dependent manner. RNA sequencing (RNA-seq) revealed SIRT1 downregulation and enrichment of related pathways. Further in vitro experiments demonstrated that aspartame inhibited SIRT1 and the PI3K/AKT/mTOR pathway, increased ROS levels, and promoted apoptosis; these effects were reversed by SIRT1 overexpression. Parallel in vivo studies showed that oral administration of aspartame to rats at 30-120 mg/kg for 8 weeks disrupted the estrous cycle, induced hormonal imbalance, caused follicular dysplasia, increased follicular atresia, and reduced ovarian reserve. Ovarian immunohistochemistry (IHC) further confirmed apoptotic damage. To further clarify the driving role of OS, assays utilizing serum from aspartame-treated mice combined with N-acetylcysteine (NAC) showed that ROS elevation and subsequent apoptosis were significantly reversed by NAC treatment. Collectively, these findings suggest that aspartame exposure contributes to POF, with direct suppression of the SIRT1/PI3K/AKT/mTOR pathway representing a key molecular mechanism underlying its intrinsic toxicity and aggravating OS-related apoptosis. This study provides mechanistic evidence for the reproductive toxicity of aspartame and supports the need for further risk assessment and exposure control to reduce the risk of POF.