Abstract
Polycystic ovary syndrome is one of the most common endocrine and metabolic gynecological disorders, of which dysfunction of ovarian granulosa cells is a key contributing factor. The aim of the present study was to explore the role of ferrostatin-1 (Fer-1), a ferroptosis inhibitor, in a cell injury model established by homocysteine (Hcy)-induced ovarian granulosa KGN cell line and the potential underlying mechanism. Cell viability was measured using Cell Counting Kit-8 assay in the presence or absence of Hcy and Fer-1. Cell apoptosis was assessed using TUNEL staining and the expression levels of apoptosis-related proteins were measured using western blotting. To explore the effects of Fer-1 on oxidative stress in Hcy-treated ovarian granulosa cells, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), lactate dehydrogenase (LDH) and glutathione (GSH) were measured using their corresponding kits. Furthermore, Fe2+ levels were assessed using Phen Green™ SK labeling and western blotting was performed to measure the protein expression levels of ferroptosis-associated proteins GPX4, SLC7A11, ASCL4 and DMT1. Subsequently, DNA methylation and ten-eleven translocation (TET) 1/2 demethylase levels were also detected to evaluate the extent of overall DNA methylation in ovarian granulosa cells after Hcy treatment. The TET1/2 inhibitor Bobcat339 hydrochloride was applied to treat ovarian granulosa cells before evaluating the possible effects of Fer-1 on TET1/2 and DNA methylation. Fer-1 was found to markedly elevate ovarian granulosa cell viability following Hcy treatment. The apoptosis rate in Fer-1-treated groups was also markedly decreased, which was accompanied by downregulated Bax and cleaved caspase-3 expression and upregulated Bcl-2 protein expression. In addition, Fer-1 treatment reduced the levels of ROS, MDA and LDH whilst enhancing the levels of GSH. Fe2+ levels were significantly decreased following Fer-1 treatment, which also elevated glutathione peroxidase 4 expression whilst reducing solute carrier family 7 member 11, achaete-scute family BHLH transcription factor 4 and divalent metal transporter 1 protein expression. Fer-1 significantly inhibited DNA methylation and enhanced TET1/2 levels, which were reversed by treatment with Bobcat339 hydrochloride. Subsequent experiments on cell viability, oxidative stress, Fe2+ content, ferroptosis- and apoptosis-related proteins levels revealed that Bobcat339 hydrochloride reversed the effects of Fer-1 on ovarian granulosa Hcy-induced cell injury. These results suggest that Fer-1 may potentially protect ovarian granulosa cells against Hcy-induced injury by increasing TET levels and reducing DNA methylation.
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