Department of Analytical Chemistry, Faculty of Pharmacy, Keio University, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2014 Apr 21;55(4):2547-56. doi: 10.1167/iovs.13-13850.
Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage.
Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis.
Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid.
Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.
干眼症（DES）的特征是泪液渗透压增加，并诱导一种具有渗透保护作用的转录因子（激活 T 细胞核因子 5 [NFAT5]）的表达和核定位，该转录因子在提供对高渗眼泪的保护方面发挥重要作用。在这项研究中，我们筛选了已在临床使用的药物，以期找到保护培养的人角膜上皮细胞免受高渗诱导的细胞损伤的化合物。
通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法测定活细胞数，并通过免疫印迹法测定细胞 NFAT5 水平。通过去除泪腺建立 DES 的大鼠模型，根据荧光素染色和上皮细胞凋亡评估角膜表面损伤。
在筛选过程中发现了一些非甾体抗炎药（NSAIDs），包括二氯芬酸钠（双氯芬酸）。这些 NSAIDs 能够抑制高渗诱导的细胞凋亡和细胞生长停滞。相比之下，其他 NSAIDs，包括溴芬酸钠（溴芬酸钠），则没有这种保护作用。在高渗条件下，用二氯芬酸处理细胞，但不用溴芬酸钠处理，可刺激 NFAT5 的核定位和表达。在 DES 的大鼠模型中，局部给予二氯芬酸（而不是溴芬酸钠）可减轻角膜表面损伤，而不影响泪液体积。
二氯芬酸似乎可保护细胞免受高渗诱导的细胞损伤，NFAT5 在这种保护作用中发挥重要作用。本研究结果还表明，局部给予二氯芬酸可能对 DES 患者具有治疗益处。
