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Jin Hyoung Park, Seong-Ho Moon, Dong Hyun Kang, Hyun Jun Um, Soon-Suk Kang, Jae Yong Kim, Hungwon Tchah; Diquafosol Sodium Inhibits Apoptosis and Inflammation of Corneal Epithelial Cells Via Activation of Erk1/2 and RSK: In Vitro and In Vivo Dry Eye Model. Invest. Ophthalmol. Vis. Sci. 2018;59(12):5108-5115. doi: https://doi.org/10.1167/iovs.17-22925.
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To evaluate the effect of diquafosol on corneal epithelium in a dry eye model using Transwell culture and a scopolamine-induced dry eye rat model.
Desiccation stress induced in an in vitro dry eye model using human corneal epithelial cells was used, and the cells were incubated with or without diquafosol media diluted at 1:100. Reactive oxygen species (ROS) generation was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was analyzed, and levels of phosphorylated Erk1/2, phosphorylated p90RSK, phosphorylated Akt, IκB-α, and NF-κB-p65 were determined. Levels of IL-1β, TNF-α, IL-6, IL-8, and GM-CSF were quantified. To investigate the in vivo effects of diquafosol, we induced dry eye in Wistar rats using scopolamine hydrobromide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol; topical DIQUAS was applied four times daily for 28 days. We used immunohistochemistry to detect the levels of phosphorylated Erk1/2, phosphorylated p90RSK, and IL-1β, and used the TUNEL assay in corneal tissue.
The distribution of highly fluorescent dichlorofluorescein and the proportion of annexin V- and PI-positive cells decreased in the diquafosol medium. Diquafosol increased the levels of phospho-Erk1/2, phospho-90RSK, phospho-Akt, and IκB-α, whereas it significantly decreased the levels of NF-κB-p65, IL-1β, and TNF-α. In vivo, apoptosis was enhanced in dry eye group. This response was markedly reduced and the level of phosphorylated p90RSK and phosphorylated ERK1/2 were upregulated and IL-1β was downregulated by DIQUAS.
Diquafosol treatment reduced intracellular ROS levels, apoptosis, and inflammation, all of which were increased in the dry eye model through desiccation.
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