PERK inhibition attenuates multi-program cell death through Nrf2/HO-1 activation in diabetic retinopathy with integrated proteomics and functional validation in HRECs

To investigate whether protein kinase R-like ER kinase (PERK) inhibition mitigates hyperglycemia (HG)-induced multi-modal cell death, including ferroptosis, apoptosis, and pyroptosis, in human retinal endothelial cells (RECs) (HRECs) through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway, thereby providing a unified therapeutic strategy for diabetic retinopathy. HRECs were cultured in normal glucose (NG, 5.5 mM, 48 h) and HG (HG, 25 mM, 48 h) DMEM media, with or without the PERK inhibitor ISRIB or the ferroptosis inhibitor Fer-1. Protein expression profiles from NG and HG-treated cells were compared using tandem mass tag (TMT)-LC-MS/MS and subjected to bioinformatic analysis. Cell viability was assessed via CCK-8 assay. Ferroptosis markers (Malondialdehyde (MDA), reactive oxygen species (ROS), Fe 2+ , GSH, glutathione peroxidase 4 (GPX4)), apoptosis mediators (caspase-3, caspase-7, Bcl-2), and pyroptosis markers (NLRP3, caspase-1, gasdermin D (GSDMD)) were quantified through biochemical assays and ELISA. Expression levels of PERK, Nrf2, and HO-1 were analyzed by real-time quantitative PCR (RT-qPCR) and Western blotting. Proteomic analysis identified 247 differentially expressed proteins (DEPs) significantly enriched in ferroptosis and peroxisome proliferator-activated receptor (PPAR) signaling pathways. HG conditions induced concurrent ferroptosis (increased MDA, ROS, Fe 2+ ; decreased ↓GSH, GPX4), apoptosis (elevated caspase-3 and caspase-7; reduced ↓Bcl-2), and pyroptosis (upregulated NLRP3, caspase-1, and GSDMD; all p < 0.001). Treatment with ISRIB reversed these effects by normalizing ferroptosis markers, suppressing apoptosis (reduced caspase-3 and caspase-7; increased ↑Bcl-2), and inhibiting pyroptosis (decreased ↓NLRP3, caspase-1, GSDMD). Mechanistically, ISRIB activated the Nrf2/HO-1 pathway while downregulating PERK, with no significant effects observed under normoglycemic conditions. PERK inhibition simultaneously attenuates HG-induced ferroptosis, apoptosis, and pyroptosis in HRECs via activation of the Nrf2/HO-1 pathway. This coordinated blockade presents a promising therapeutic strategy to preserve retinal vasculature in diabetic retinopathy.