Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China; NHC Key Laboratory of Myopia, Fudan University, Shanghai, China; Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China.
Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China; NHC Key Laboratory of Myopia, Fudan University, Shanghai, China; Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China.
Exp Eye Res. 2020 Nov;200:108210. doi: 10.1016/j.exer.2020.108210. Epub 2020 Sep 5.
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.
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 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.
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.
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.
此前，已有研究表明骨化三醇是干眼症的一种潜在治疗药物，但其对角膜上皮细胞死亡的作用尚不清楚。本研究旨在探讨凋亡和自噬在干眼症相关情况下的关系，以及骨化三醇的作用及其潜在机制。
在体外，将永生化人角膜上皮细胞（iHCEC）培养在高渗培养基中，或在高渗培养基中加入不同浓度的骨化三醇和其他试剂。在体内，应用苯扎氯铵诱导 Wistar 大鼠干眼症，然后用骨化三醇（10 μM）局部治疗 14 天。通过 Western blot 或免疫染色检测自噬流（LC3B-II 和 SQSTM1/P62）。通过 Western blot 检测细胞凋亡，用 Annexin V/PI 双染和 TUNEL 检测法检测细胞凋亡，用 CCK-8 检测细胞活力。用小干扰 RNA 敲低 iHCECs 中的维生素 D 受体表达。
自噬激活可保护 iHCECs 免受 HS 诱导的体外细胞凋亡，骨化三醇通过 VDR 信号通路增强自噬流，表现为 LC3B-II 表达显著增加，p62 表达降低。体内结果进一步支持了骨化三醇通过促进自噬在干眼症大鼠中预防角膜上皮细胞凋亡的保护作用。
本研究表明，自噬是角膜上皮细胞对高渗应激的适应性变化，骨化三醇通过 VDR 途径进一步激活自噬，可防止细胞凋亡。
