Palmitic acid enhances the sensitivity of ferroptosis via endoplasmic reticulum stress mediated the ATF4/TXNIP Axis in polycystic ovary syndrome

BACKGROUND: Palmitic acid (PA), the most prevalent saturated fatty acid in humans, is closely associated with ovarian dysfunction. Elevated PA levels in the follicular fluid of patients with polycystic ovary syndrome (PCOS) are correlated with the outcomes of assisted reproductive technology (ART), though the underlying mechanism remains unclear. METHODS: Multi-omics analysis identified PA and TXNIP as potential pathogenic factors in PCOS. CCK-8 and apoptosis assay were conducted to detect the cytotoxicity of PA. To further investigate the molecular mechanisms underlying PA-induced ferroptosis, we established COV434 cell models with both TXNIP overexpression and knockdown. Transmission electron microscopy (TEM), western blot (WB), ELISA assays, and flow cytometry were employed to assess ferroptosis-related markers. A PCOS mouse model was also developed, and histopathological staining, TEM, ELISA, and WB were performed to evaluate clinical parameters related to PCOS ovarian ferroptosis levels. To clarify the targeting relationship between ATF4 and TXNIP, we utilized luciferase reporter gene assays, chromatin immunoprecipitation (ChIP), and RT-qPCR for a comprehensive analysis. RESULTS: In vivo and in vitro, PA enhanced the sensitivity of PCOS ovarian ferroptosis. The protein levels of TXNIP and ACSL4 were upregulated in both PCOS patients and mouse models after PA treatment. PA also induces the expression of the ferroptosis inhibitor SLC7A11 as part of an adaptive response. Elevated intracellular ROS levels, increased MDA content, decreased GSH/GSSG ratios, elevated ferrous iron levels, and TEM findings collectively indicated that PA induces ferroptosis in KGN/COV434 cells. The ER stress inhibitor 4-PBA reduces PA-induced ferroptosis in PCOS ovaries by suppressing ER stress, thereby improving PA-induced PCOS-like traits. Moreover, the UPR gene ATF4 regulates cellular ferroptosis by activating the transcriptional expression of TXNIP. CONCLUSION: PA stimulated ovarian ferroptosis by activating ER stress, a process mediated by the ATF4/TXNIP axis, which might represent a potential mechanism underlying the progression of PCOS. The application of ER stress inhibitors improved PCOS traits by reducing the sensitivity of ovarian ferroptosis.