UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells.
UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells.
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DOI：
10.1167/iovs.16-19097
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发表时间：
2016-04-01
影响因子：
4.4
通讯作者：
Jurkunas UV
中科院分区：
文献类型：
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作者：
Liu C;Vojnovic D;Kochevar IE;Jurkunas UV
目的：检测环境水平的紫外线A(一种已知的氧化应激刺激因子)是否激活了Nrf2调节的人角膜内皮细胞(CEnCs)中的抗氧化防御和P53。永生化的人CEnCs(HCEnCi)暴露于2.5、5、10或25J/cm2的UV-A剂量，然后允许恢复3-24小时。对照HCEnCi未接受UV-A照射。用H_2DCFDA测定细胞内活性氧(ROS)。乳酸脱氢酶(LDH)释放法检测细胞毒性。免疫印迹法或实时定量聚合酶链式反应检测Nrf2、HO-1、NQO-1、P53和caspase3的表达水平。免疫印迹和荧光分析检测激活的caspase3。与对照组相比，HCEnCi暴露于5、10和25J/cm2的UV-A可增加ROS水平。2.5和5J/cm2 UV-A照射后3h和6h，NRF2、HO-1和NQO-1mRNA表达增加1.7~3.2倍。照射后6h，UV-A(5J/cm2)可促进Nrf2核转位。在治疗24小时后，UV-A(5、10和25J/cm2)产生的磷酸化P53是对照组的1.8到2.8倍，激活的caspase3是对照组的2.6到6.0倍，导致20%到42%的细胞死亡。低剂量的UV-A诱导Nrf2调节的抗氧化防御，高剂量的UV-A激活P53和caspase3，表明即使是接近环境水平的UV-A也可能影响正常的CEnc。这一数据表明，UV-A可能尤其会损伤缺乏抗氧化防御的细胞，因此可能参与了Fuchs内皮性角膜营养不良(FECD)的病因。
To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).