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Molecular medicine reports

Molecular medicine reports

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Ferroptosis inhibitor ferrostatin?1 alleviates homocysteine?induced ovarian granulosa cell injury by regulating TET activity and DNA methylation

Published:1 April 2022 DOI: 10.3892/mmr.2022.12645 PMID: 35169856
Qing Shi, Rui Liu, Li Chen

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.

Substances (4)

Materials
Procduct Name CAS Molecular Formula Supplier Price
Bobcat339 (Bobcat 339) 2280037-51-4 C16H12ClN3O 54 suppliers Inquiry
Bobcat339 (Bobcat 339) 2280037-51-4 C16H12ClN3O 54 suppliers Inquiry
Bobcat339 (Bobcat 339) 2280037-51-4 C16H12ClN3O 54 suppliers Inquiry
Bobcat339 (Bobcat 339) 2280037-51-4 C16H12ClN3O 54 suppliers Inquiry

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