HYPEROSMOTIC STRESS INDUCES PANOPTOSIS OF CORNEAL EPITHELIAL CELLS AND INCREASED EXPRESSION OF INFLAMMATORY FACTORS BY ACTIVATING ZBP1

Abstract

Background: Dry eye disease (DED) is closely associated with structural damage and dysfunction of the ocular surface, particularly affecting the corneal epithelium. A commonly used in vitro model to study this damage involves exposing corneal epithelial cells to hypertonic saline, simulating the hyperosmotic stress observed in DED. Previous studies have shown that multiple forms of programmed cell death contribute to corneal damage in DED. Understanding the role of ZBP1 in mediating PANoptosis—a collective term encompassing pyroptosis, apoptosis, and necroptosis—during hyperosmotic stress is crucial for gaining further insights into the mechanisms of ocular surface damage in DED. Objective: This study aims to investigate whether PANoptosis is involved in the programmed death of corneal epithelial cells under hyperosmotic stress and to explore the regulatory role of ZBP1 in hyperosmotic-induced corneal damage. Method: Corneal epithelial cells were cultured under hypertonic saline conditions to simulate hyperosmotic stress. ZBP1 expression was knocked down using ZBP1-siRNA. Cell morphology, viability, and migration were assessed, while apoptosis and inflammatory mediator expression were evaluated through qPCR and Western blotting. Results: We found that corneal epithelial cells exposed to increasing osmolarities (400, 450, and 500 mOsm) exhibited progressive cell death and damage. The predominant mode of cell death varied with osmolarity: apoptosis was the primary mechanism at 400 mOsm, whereas pyroptosis and necroptosis became more prominent at 450 mOsm. Hyperosmotic stress also led to significant reductions in cell viability and migration, along with marked upregulation of ZBP1 and PANoptosis-related cytokines and inflammatory markers. Importantly, we found that silencing ZBP1 using siRNA improved the cells' tolerance to hyperosmotic stress, reducing both cell death markers and the expression of inflammatory mediators. Conclusions: Our findings suggest that increasing osmolarity exacerbates corneal epithelial cell death and inflammation, largely through PANoptosis pathways. ZBP1 upregulation plays a pivotal role in mediating hyperosmotic-induced corneal damage. Silencing ZBP1 effectively reduces PANoptotic cell death and inflammation, highlighting its potential as a therapeutic target for treating dry eye disease.