Effect of High Glucose on Ocular Surface Epithelial Cell Barrier and Tight Junction Proteins
Effect of High Glucose on Ocular Surface Epithelial Cell Barrier and Tight Junction Proteins
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DOI：
10.1167/iovs.61.11.3
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发表时间：
2020-09-01
影响因子：
4.4
通讯作者：
Sharma, Ajay
中科院分区：
文献类型：
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作者：
Alfuraih, Saleh;Barbarino, Ashley;Sharma, Ajay
目的。据报道，糖尿病患者存在眼表缺陷、眼表屏障功能受损以及角膜和结膜感染的发生率较高。紧密连接对于眼表屏障功能至关重要。本研究旨在探讨高糖暴露对人角膜和结膜上皮细胞屏障功能和紧密连接蛋白的影响。方法。人角膜和结膜上皮细胞分别暴露于 15 mM 和 30 mM 葡萄糖中 24 和 72 小时。使用跨上皮电阻（TEER）测量屏障功能。使用划痕测定对细胞迁移进行定量。收获细胞用于蛋白质提取和 mRNA 分离。使用实时 PCR 和蛋白质印迹对 Claudins、闭合小带 (ZOs) 和 Occludin 的基因和蛋白表达进行定量。 结果。接触葡萄糖 72 小时后，角膜和结膜上皮细胞的 TEER 显着下降。葡萄糖不会引起角膜或结膜上皮细胞迁移的任何显着变化。葡萄糖暴露不会导致claudin-1、ZO-1、ZO-2、ZO-3或occludin的蛋白质表达发生任何显着变化。另一方面，15 mM 葡萄糖导致claudin-1、claudin-3、ZO-2、ZO-3 和occludin 的基因表达增加，这可能是对渗透压的反应，因为15 mM 甘露醇也导致这些蛋白质的基因表达持续相似的增加。结论。高葡萄糖暴露会导致角膜和结膜上皮细胞屏障功能受损，但这种有害影响并不是由紧密连接蛋白（claudin-1、ZO-1、ZO-2、ZO-3 和 occludin）表达减少引起的。
PURPOSE. Patients with diabetes mellitus are reported to have ocular surface defects, impaired ocular surface barrier function, and a higher incidence of corneal and conjunctival infections. Tight junctions are critical for ocular surface barrier function. The present study was designed to investigate the effect of high glucose exposure on human corneal and conjunctival epithelial cell barrier function and tight junction proteins.METHODS. Human corneal and conjunctival epithelial cells were exposed to 15 mM and 30 mM glucose for 24 and 72 hours. The barrier function was measured using transepithelial electrical resistance (TEER). The cell migration was quantified using scratch assay. The cells were harvested for protein extraction and mRNA isolation. Gene and protein expression of claudins, zonula occludens (ZOs), and occludin was quantified using real-time PCR and Western blot.RESULTS. Glucose caused a significant decrease in TEER after 72 hours of exposure in both corneal and conjunctival epithelial cells. Glucose did not cause any notable change in migration of either corneal or conjunctival epithelial cells. Glucose exposure did not cause any notable change in protein expression of claudin-1, ZO-1, ZO-2, ZO-3, or occludin. On the other hand, 15 mM glucose caused an increase in gene expression of claudin-1, claudin-3, ZO-2, ZO-3, and occludin, a likely response to osmotic stress since 15 mM mannitol also caused consistently similar increase in gene expression of these proteins.CONCLUSIONS. High glucose exposure causes impairment of corneal and conjunctival epithelial cell barrier function, but this detrimental effect is not caused by a decrease in expression of tight junction proteins: claudin-1, ZO-1, ZO-2, ZO-3, and occludin.