Abstract
Purpose :
Exposure keratopathy (EK) is damage to the cornea that occurs primarily from prolonged exposure of the ocular surface to the outside environment. In this study, we investigated the role of autophagy in response to air exposure injury in EK.
Methods :
EK models were constructed by human corneal epithelial cells (HCECs) and C57BL/6 mice respeatively. Transmission electron microscopy (TEM) was used to detect changes of corneal epithelial cells after air exposure. Air exposured HCEs and mice were treated with autophagy inhibitors/activators, chloroquine diphosphate (CQ) or Rapamycin (Rap). Autophagy-related proteins, LC3B, SQSTM1/p62, Beclin1 and mTOR, were assassed by immunohistochemistry. Western blotting was used to detect the expressions of endoplasmic reticulum (ER) stress-related proteins (PERK, eIF2α, and CHOP), PI3K/Akt/mTOR signaling pathway-related proteins. The exppression of inflammation-related factors IL1-β, IL-6 and MMP9 were analyzed by qRT-PCR. TUNEL assay was applied to detect and quantitate apoptotic cells.
Results :
We observed autophagic vacuoles in air-exposured HCECs and mice by TEM. Autophagy induction was further confirmed by increased LC3 puncta and decreased p62 by immunofluorescence staining and western blotting. Increased TUNEL positive staining were detected in CQ treated HCECs, while Rap treatment resulted in a reduced number of apoptotic cells. Similarly, CQ injection enhances air exposure-induced apoptosis and inflammation in mouse corneal epithelium, which were inhibited by Rap treatment. Furthermore, western blot assay showed that phosphorylation of PERK and eIF2α and CHOP expression increased after air exposure, indicating that ER stress- autophagy activated for cell survival after corneal air exposure injury.
Conclusions :
Autophagy can be induced by air-exposed injury, and autophagy is indispensable for the maintenance of ocular physiology and corneal epithelial cell survival.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.