Abstract
Purpose :
Until now, little is known about the change of autophagy and its underlying mechanism in dry eye development. Here we hypothesize that reactive oxygen species(ROS) elevation through induction of the metabolic regulator DDIT4 is transduced to impair autophagy in dry eye disease(DED).
Methods :
Human corneal epithelial cells(HCECs) were exposed to hyperosmolar media, C57BL/6 mice were given subcutaneous scopolamine hydrobromide injections to imitate experimental dry eye(EDE) in vitro and in vivo. Western blot was used to measure the change of protein expression such as LC3-II, ATG-5, Beclin1, SQSTM1, DDIT4, Caspase-3, etc. Autophagosome was observed by electron microscopy(EM). HCECs were transfected with siRNA to knockdown DDIT4. Immunostaining was applied to indicated autophagy flux. Cellular ROS and mitochondrial function were detected by flow cytometry. Cell Counting Kit-8 assay and LDH assay were used to measure cell death. The change of inflammatory cytokines was verified by qPCR.
Results :
Under EDE, western blot analysis showed a significant increase of LC3-II both in vitro and in vivo, with slight change of BECN1 and ATG5. Furthermore, we found SQSTM1, a molecule mainly degraded through autophagy, accumulated in a time-dependent fashion. Besides, EM showed large autophagic vacuoles with poorly degraded material in corneal epithelium. Meanwhile, western blot analysis showed an elevated expression of DDIT4 and inhibition of mTOR pathway under hyperosmolarity. Interestingly, when DDIT4 expression was blocked with siRNA transfection, we observed a decreased levels of LC3-II, SQSTM1, along with normalized ROS level and mitochondrial function. In order to further investigate the crosstalk between ROS and autophagy, A ROS scavenger, N-acetylcysteine (NAC) was used. Our results indicated that NAC could also reduce the level of LC3-II and SQSTM1. Lastly, we discovered that DDIT4 knockdown efficiently protected cell viability, inhibited apoptosis and attenuated inflammation cytokins release under hyperosmolar stress.
Conclusions :
Our research for the first time suggests that the DDIT4-mediated ROS signaling pathways could lead to impaired autophagy. Recognition of a DDIT4-ROS-autophagy axis involved in dry eye development may shed a light upon future understanding of the pathogenesis in DED and thereby help to innovate new therapeutic interventions.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.