3D cultured human umbilical cord mesenchymal stem cell spheroids regulate oxidative stress and iron homeostasis through the Nrf2 pathway to resist ferroptosis in ovarian granulosa cells and ovarian dysfunction.

Oxidative stress-induced death of ovarian granulosa cells (GCs) is a major driver of ovarian functional disorders associated with follicular atresia. Ferroptosis is a key factor in the onset and progression of various ovarian oxidative stress-related diseases, making it a potential target for enhancing reproductive health. Recently, 3D cultured human umbilical cord mesenchymal stem cells (3D hUCMSCs) spheroids have exhibited promising advantages in protecting GCs from oxidative damage. However, it is unclear whether they represent a viable therapeutic strategy for mitigating reproductive failure associated with abnormal follicular atresia by modulating ferroptosis. This study demonstrated that 3D hUCMSC spheroids can effectively protect GCs from hydrogen peroxide (H2O2)-induced oxidative stress and ferroptosis. Additionally, iron overload and lipid peroxidation are two essential features of ferroptosis. 3D hUCMSC spheroids effectively regulate iron uptake and storage to mitigate H2O2-induced iron overload. Furthermore, 3D hUCMSC spheroids mitigate lipid peroxidation induced by H2O2 by restoring GSH metabolic balance and preventing GPX4 inactivation. Mechanistically, the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway was significantly activated by 3D hUCMSC spheroid treatment. Our findings reveal that Nrf2 knockdown inhibited the 3D hUCMSC spheroids-mediated resistance of GCs to H2O2-induced ferroptosis, and Nrf2 knockdown led to increased iron uptake, resulting in substantial lipid peroxidation through the Fenton reaction, thereby making GCs more susceptible to ferroptosis. This process may involve the ROS-Nrf2-Fe2+ cycle. Significantly, 3D hUCMSC spheroids can mitigate H2O2-induced ferroptosis in GCs by regulating the ROS-Nrf2-Fe2+ cycle. Finally, we confirmed the above results that 3D hUCMSC spheroids ameliorate ovarian oxidative damage in premature ovarian failure (POF) rats. In conclusion, we demonstrated that 3D hUCMSC spheroids regulate oxidative stress and iron homeostasis through the Nrf2 pathway, thereby providing a potential therapeutic target for anovulatory disorders.