June 2020
Volume 61, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2020
Calcitriol Inhibits Apoptosis via Activation of Autophagy in Hyperosmotic Stress Stimulated Corneal Epithelial Cells in vivo and vitro
Author Affiliations & Notes
  • Ning Lyu
    Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai, Shanghai, China
  • Jing Zhang
    Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai, Shanghai, China
  • Yiqin Dai
    Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai, Shanghai, China
  • Jun Xiang
    Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai, Shanghai, China
  • Yue Li
    Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai, Shanghai, China
  • Jianjiang Xu
    Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Medical College of Fudan University, Shanghai, Shanghai, China
  • Footnotes
    Commercial Relationships   Ning Lyu, None; Jing Zhang, None; Yiqin Dai, None; Jun Xiang, None; Yue Li, None; Jianjiang Xu, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 139. doi:
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      Ning Lyu, Jing Zhang, Yiqin Dai, Jun Xiang, Yue Li, Jianjiang Xu; Calcitriol Inhibits Apoptosis via Activation of Autophagy in Hyperosmotic Stress Stimulated Corneal Epithelial Cells in vivo and vitro. Invest. Ophthalmol. Vis. Sci. 2020;61(7):139.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Apoptosis induced by tear hyperosmolarity is the main cause of corneal epithelial cell death, which further leads to clinical symptoms in dry eye. Moreover, the role of crosstalk between apoptosis and autophagy in dry eye still remains unclear. Calcitriol, the active metabolite of vitamin D3, was previously reported to alleviate the dry eye symptoms. We hypothesize that autophagy plays an adaptive role against apoptosis induced by hyperosmolarity, through which calcitriol can protect corneal epithelial cells.

Methods : The immortalized human corneal epithelial cells (iHCECs) were cultured with or without hyperosmotic stress (HS), calcitriol (Cal) and other reagents. Rapamycin (RAPA) or bafilomycin A1 (Bafi) was used to induce or block autophagy. Autophagy was evaluated by western blot for LC3B and P62 and immunostaining of LC3B. To test apoptosis, CCK-8 assay, western blot for cleaved caspase-3 and flow cytometer for Annexin V-FITC/PI were used. siRNA was used to knockdown the gene expression of vitamin D receptor (VDR). Wistar rats were treated by benzalkoniumchloride (BAC) bid for 14 days in dry eye group. Then rats in treatment group were treated with calcitriol tid for 10 days. PBS was applied as control. Apoptosis of rat corneal epithelial cells was examined by TUNEL staining.

Results : The highest expression of LC3B, lowest expression of P62 and highest positive LC3B staining rate were observed in Cal and HS group followed by HS gourp and control group. By using RAPA, it showed that both expression of cleaved caspase-3 and apoptotic incidence were reduced, while the cell viability was rose. Similarly, Cal contrary to Bafi indicated the same results. In addition, the combination of RAPA and Cal had a synergistic effect on anti apoptosis. The knockdown of VDR attenuated autophagy and anti-apoptosis effect induced by Cal. In vivo, the treatment group had the lowest expression of P62, highest expression of LC3B and strongest fluorescence intensity of LC3B staining. TUNEL staining showed that the apoptotic incidence of corneal epithelial cells in treatment group was lower than dry eye group.

Conclusions : These findings showed that autophagy was an adaptive alteration for corneal epithelial cells to hyperosmolarity-induced apoptosis and subsequently, calcitriol can protect cells from apoptosis through further activation of autophagy, which fits our hypothesis.

This is a 2020 ARVO Annual Meeting abstract.

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