Calcitriol inhibits apoptosis via activation of autophagy in hyperosmotic stress stimulated corneal epithelial cells in vivo and in vitro.

BACKGROUND

Previously, calcitriol has been demonstrated as a potential therapeutic agent for dry eye, whilst its role on corneal epithelium death remains unclear. This study aims to investigate the relationship between apoptosis and autophagy on dry eye related scenario, as well as the effect of calcitriol and its potential mechanism.

METHODS

In vitro, immortalized human corneal epithelial cells (iHCEC) were cultured in hyperosmotic medium with or without various concentrations of calcitriol and other reagents. In vivo, Wistar rats were applied with benzalkonium chloride to induce dry eye. Then rats were topically treated with calcitriol (10-6 M) for 14 days. Autophagy flux (LC3B-II and SQSTM1/P62) was examined by western blotting or immunostaining. To test cell apoptosis, western blotting for cleaved caspase-3, Annexin V/PI double staining and TUNEL assay were used. CCK-8 assay was performed to detect the cell viability. Small interfering RNA was used to knock down the expression of vitamin D receptor in iHCECs.

RESULTS

Autophagy activation could protect iHCECs against HS induced apoptosis in vitro, and calcitriol was able to augment autophagy flux via VDR signaling, shown as the remarkably elevated expression of LC3B-II, as well as the declined p62 expression. In vivo results further supported the protective role of calcitriol on corneal epithelium apoptosis through promoting autophagy in dry eye rats.

CONCLUSION

The current study indicated that autophagy was an adaptive change of corneal epithelial cells in response to hyperosmotic stress and calcitriol could prevent cells from apoptosis via further activation of autophagy through VDR pathway.