Metformin inhibits ovarian granular cell pyroptosis through the miR-670-3p/NOX2/ROS pathway

Recent studies have demonstrated that ovarian granular cells (OGCs) pyroptosis is present in the ovaries of polycystic ovary syndrome (PCOS) mice and that NLRP3 activation destroys follicular functions. Metformin has been shown to protect against PCOS by reducing insulin resistance in women, whereas its role in OGC pyroptosis is unknown. This study aimed to investigate the impact of metformin on OGC pyroptosis and the underlying mechanisms. The results showed that treating a human granulosa-like tumor cell line (KGN) with metformin significantly decreased LPS-induced expression of miR-670-3p, NOX2, NLRP3, ASC, cleaved caspase-1, and GSDMD-N. Cellular caspase-1 activity; ROS production; oxidative stress; and the secretion of IL-1β, IL-6, IL-18, and TNF-α were also diminished. These effects were amplified by adding N-acetyl-L-cysteine (NAC), a pharmacological inhibitor of ROS. In contrast, metformin's anti-pyroptosis and anti-inflammatory effects were robustly ameliorated by NOX2 overexpression in KGN cells. Moreover, bioinformatic analyses, RT-PCR, and Western blotting showed that miR-670-3p could directly bind to the NOX2 (encoded by the CYBB gene in humans) 3'UTR and decrease NOX2 expression. Metformin-induced suppression of NOX2 expression, ROS production, oxidative stress, and pyroptosis was significantly alleviated by transfection with the miR-670-3p inhibitor. These findings suggest that metformin inhibits KGN cell pyroptosis via the miR-670-3p/NOX2/ROS pathway.