Growth Arrest-specific 1 Inhibits Keap1/Nrf2 Signaling Transduction in the Activation of the Ferroptosis Program in Retinal Müller Cells.

Abstract

Background: Diabetes retinopathy (DR) represents a microvascular disease in diabetes. Growth arrest-specific 1 (GAS1) is differentially expressed in rat retinal Müller cells under high glucose (HG) conditions, and its promotion of ferroptosis contributes to retinal cell death. However, the influence of GAS1 in DR is elusive. Herein, we aimed to investigate the effect and potential mechanism based on GAS1-mediated ferroptosis on DR.

Methods: After HG treatment, the differentially expressed genes in rat retinal Müller cells were analyzed by transcriptome sequencing followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses; finally, GAS1 was selected. The effects of GAS1 knockdown/overexpression and nuclear factor erythroid 2-related factor (Nrf2) silencing on viability, apoptosis, lipid peroxidation, Fe²⁺, and oxidative stress levels in HG-induced/transfected Müller cells were measured by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and commercial reagent kits. The potential effects of GAS1 and Nrf2, especially on GAS1, Nrf2, and Kelch-like ECH-associated protein 1 (Keap1) expressions in cells, were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot.

Results: HG treatment decreased cell viability and glutathione (GSH) levels and increased apoptosis, lipid reactive oxygen species (ROS), glutathione disulfide (GSSG), malondialdehyde (MDA), oxidative stress, and Fe²⁺ levels in Müller cells (p < 0.01). HG treatment also upregulated GAS1, Keap1, and total Nrf2 expressions while downregulating nuclear Nrf2 in Müller cells (p < 0.001). GAS1 downregulation enhanced cell viability, GSH levels, and nuclear Nrf2 expression while reducing the levels of apoptosis, lipid ROS, GSSG, MDA, Fe²⁺, Keap1, and total Nrf2 in HG-treated Müller cells (p < 0.001), whereas GAS1 overexpression had the opposite effects. Additionally, Nrf2 silencing reversed the impact of GAS1 overexpression in HG-treated Müller cells (p < 0.05).

Conclusion: GAS1 inhibits Keap1/Nrf2 signaling transduction in activating ferroptosis in retinal Müller cells; thus, this study can aid in setting the stage for novel treatment methods against DR.