Endoplasmic Reticulum Stress Induces ROS Production and Activates NLRP3 Inflammasome Via the PERK-CHOP Signaling Pathway in Dry Eye Disease

Abstract

PURPOSE. The purpose of this study was to investigate the potential roles of endoplasmic reticulum (ER) stress in the development of dry eye disease (DED). METHODS. Single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, derived from corneal tissues of a dry eye mouse model, was processed using the Seurat R program. The results were validated using a scopolamine-induced dry eye mouse model and a hyperosmotic-induced cell model involving primary human corneal epithelial cells (HCECs) and immortalized human corneal epithelial (HCE-2) cells. The HCE-2 cells were treated with 4-phenylbutyric acid (4-PBA) or tunicamycin (TM) to modulate ER stress. TXNIP and PERK knockdown were performed by siRNA transfection. Immunofluorescence, Western blotting, and real-time PCR were used to assess oxidative stress, ER stress, unfolded protein response (UPR) marker proteins, and TXNIP/NLRP3 axis activation. RESULTS. The analysis of scRNAseq data shows an increase in the ER stress marker GRP78, and the activation of the PERK-CHOP of UPR in DED mouse. These findings were confirmed both in vivo and in vitro. Additionally, HCE-2 cells treated with 4-PBA or TM showed significant effects on the production of reactive oxygen species (ROS) and the activation of the TXNIP/NLRP3-IL1β signaling pathway. Furthermore, siRNA knockdown of PERK or TXNIP, which alleviated the TXNIP/NLRP3-IL1β signaling axis, showed protective effects on HCECs. CONCLUSIONS. This study explores the role of ER stress-induced oxidative stress and NLRP3-IL-1β mediated inflammation in DED, and highlights the therapeutic potential of PERK-CHOP axis and TXNIP in the treatment of DED.