Abstract
Purpose :
To evaluate the role of lipid droplets (LDs) in the cell fate of corneal epithelial cells and explore the underlying mechanisms in dry eye disease (DED).
Methods :
C57BL/6 mice were injected with scopolamine subcutaneously and an immortalized human corneal epithelial cell line (HCEC) was cultured with the hypertonic medium to establish DED models. The small interfering RNA (Si-RNA) modified with cholesterol was subconjunctival injected into the mice for Atg5 inhibition in vivo. Si-RNA and recombinant lentiviral were used to regulate the Atg5 gene expression in HCECs. BODIPY 493/503 fluorescence probe and Lysotracker Red DND-99 were used to label cellular LDs and lysosomes respectively. Protein expressions were measured using western blot assay and immunofluorescence staining. The intracellular ultrastructure was observed with transmission electron microscopy. The content of cellular free fatty acid was detected by a microplate reader. Flow cytometry was used to evaluate cellular lipid peroxidation.
Results :
LDs were decreased in the corneal epithelial cells in DED models with the reduced expression of LDs-associated proteins. The RNA-sequencing analysis showed that the degradation pathway was activated obviously. Specifically, we supposed lipophagy was responsible for the catabolism of LDs, as indicated by the increased co-localization of lysosomes and LDs and the autophagic vacuoles containing LDs. Moreover, the autophagy-related gene Atg5 was upregulated to mediate the degradation of LDs via binding with LD-associate protein perilipin 3. When Atg5 was knocked down, the cellular LDs increased, thereby accompanied by a reduced cellular free fatty acid, lower lipid peroxidation levels, increased cell viability, and a marked downregulation in ferroptosis markers, whereas overexpression of Atg5 produced the opposite results. It was observed consistently that Atg5 inhibition reversed corneal epithelial defects significantly in the DED mouse model.
Conclusions :
Atg5 was upregulated to mediate lipophagy, which induced LDs catabolism in corneal epithelial cells in DED, eventually leading to the occurrence of ferroptosis, and promoting corneal defects in DED. Hence, these findings revealed the role of Atg5-mediated lipophagy in DED pathogenesis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.