Protection of the cornea from UVB radiation by elevated potassium in tears

通过提高泪液中的钾含量来保护角膜免受 UVB 辐射

基本信息

批准号：
8574193
负责人：
JOHN L UBELS
金额：
$ 37.59万
依托单位：
CALVIN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2017-09-29
项目状态：
已结题

项目摘要

Project summary The tears, for the most part, have an electrolyte composition like other extracellular fluids. The tears are unique, however, in that the K+ concentration ([K+] is about 20-25 mM, compared to about 5 mM in other extracellular fluids. Exposure to ultraviolet radiation causes apoptosis of cells. UVB radiation at doses relevant to ambient levels of outdoor UVB exposure activates K+ channels leading to rapid loss of intracellular K+ (K+i). We have shown that incubation of corneal epithelial cells in medium with 25 mM K+ after exposure to UVB prevents loss of K+i and inhibits activation of apoptotic mechanisms. These observations support our overall hypothesis that the high concentration of potassium ions in tears contributes to the protection of the corneal epithelium from ambient UVB-induced damage by reducing the loss of intracellular K+ when channels are activated by UVB. We now propose to investigate the intracellular signaling mechanisms by which UVB activates K+ channels and apoptotic signaling in corneal epithelial cells. The specific aims of the proposed research are: 1) to elucidate the relationships between Fas ligand- independent, UVB-induced activation of apoptotic signaling pathways and activation of K+ channels. This will be accomplished by studying the effects of UVB on apoptotic pathways and K+ channel activation in corneal epithelial cells after siRNA knockdown of Fas and FADD, or inhibition of caspase-8; 2) To investigate whether UVB-induced K+ channel activation is the primary event in activation of apoptotic pathways. K+ channels in corneal epithelial cells will be identified using a PCR array. siRNA will then be used to knock down specific channels. This will be followed by investigation of UVB-induced activation of apoptosis, as well as effects of UVB on K+ currents and intracellular [K+] when specific channels have been knocked down; 3) Using an isolated pig eye model, effects of UVB on the intact corneal epithelium will be investigated in a configuration that allows for superfusion of the ocular surface with physiologic saline in a manner that models the bathing of the cornea with tears. The hypothesis will be tested that elevated [K+] will protect the superficial corneal cell layers from UV-induced apoptosis.

项目摘要泪水在大多数情况下具有像其他细胞外流体一样具有电解质成分。眼泪但是，独特的是，K+浓度（[K+]约为20-25毫米，而其他约5毫米细胞外流体。暴露于紫外线辐射会导致细胞凋亡。剂量的UVB辐射与环境水平相关的室外UVB暴露会激活K+通道，导致细胞内快速丧失 K+（K+ I）。我们已经表明，暴露于25 mm K+的角膜上皮细胞暴露后 UVB防止K+I的损失，并抑制凋亡机制的激活。这些观察支持我们总体假设，泪水中高钾离子的浓度有助于保护通过降低通道时的细胞内K+的损失，来自环境UVB诱导的损伤的角膜上皮被UVB激活。现在，我们建议研究UVB的细胞内信号传导机制激活角膜上皮细胞中的K+通道和凋亡信号传导。拟议研究的具体目的是：1）阐明FAS配体之间的关系独立的UVB诱导的凋亡信号通路的激活和K+通道的激活。这会可以通过研究UVB对角膜中凋亡途径和K+通道激活的影响来完成 Fas和FADD siRNA敲低后的上皮细胞，或抑制caspase-8； 2）调查是否 UVB诱导的K+通道激活是激活凋亡途径的主要事件。 K+通道中角膜上皮细胞将使用PCR阵列鉴定。然后，siRNA将用于拆除特定频道。随后将研究UVB诱导的凋亡激活，以及当特定通道被击倒时，在K+电流上的UVB和细胞内[K+]； 3）使用孤立的猪眼模型，UVB对完整角膜上皮的影响将以构型进行研究这允许以生理盐水为模型的生理盐水对眼表面进行超级灌注泪水的角膜。该假设将检验，升高[K+]将保护表面角膜紫外线引起的细胞凋亡的细胞层。