Impairment of conjunctival glutathione secretion and ion transport by oxidative stress in an adenovirus type 5 ocular infection model of pigmented rabbits.
Impairment of conjunctival glutathione secretion and ion transport by oxidative stress in an adenovirus type 5 ocular infection model of pigmented rabbits.
复制标题
色素兔腺病毒 5 型眼部感染模型中氧化应激对结膜谷胱甘肽分泌和离子转运的损害。
DOI：
10.1016/j.freeradbiomed.2004.04.030
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发表时间：
2004
影响因子：
7.4
通讯作者：
Lee,VincentHL
中科院分区：
文献类型：
--
作者：
Kannan,Ram;Gukasyan,HovhannesJ;Zhang,Wenzheng;Trousdale,MelvinD;Kim,Kwang-Jin;Lee,VincentHL
色素兔的结膜上皮细胞分泌还原型谷胱甘肽（GSH）进入顶端（粘膜）液。本研究的目的是确定病毒感染引起的氧化应激和补充GSH对氧化还原状态，GSH和离子转运在新鲜切除的结膜组织和上皮细胞层的原代培养物（RCEC）的腺病毒5型（Ad 5）感染的兔的效果。脂质过氧化（LPO）产物，一氧化氮（NO），一氧化氮合酶（NOS 2）的表达作为病毒接种后时间的函数进行定量。还测量了[3 H]GSH的单向通量和用GSH和谷胱甘肽单乙酯（GSH-β）补充Ad 5接种的结膜组织的粘膜短路电流（Isc）的变化。在感染后8、24、48和72 h，Ad 5接种使结膜GSH水平分别显著降低19、45、48和50%。在Ad 5接种后第3天，LPO产物和NO水平显著增加（分别为未感染对照的2倍和100倍），并且用GSH-β 2和生育酚琥珀酸酯共处理消除了这种效应。NO水平在Ad 5接种后显示出进行性增加，分别在第1、3和5天达到0.22 ± 0.06、8.12 ± 0.91和2.05 ± 0.65 μM，并且在病毒最大复制当天（第3天）观察到最高水平。在Ad 5接种后第1、3和5天观察到非常显著的NOS 2表达诱导。未感染的对照结膜组织显示出净浆液-粘膜GSH通量（Jsm），其中粘膜-浆液通量（Jms）为1.14 pmol h−1cm− 2，Jsm为1.22 pmol h−1cm−2。在Ad 5接种的兔中，在sm和ms方向上观察到类似的GSH通量，并且净GSH通量可以忽略不计。与对照组相比，Ad 5接种组兔结膜组织的跨膜电位差（PD）降低≥50%，而跨上皮电阻（TEER）保持不变。在Ad 5接种的结膜组织中，粘液灌注GSH或GSH-β在100 min内使Iscby增加了40%。我们的结果表明，Ad 5接种后，兔结膜GSH的净分泌被完全阻断，活性离子转运率下降了约50%。Ad 5感染后进入粘膜液的净GSH分泌减少可能是由于氧自由基诱导的氧化还原状态变化和活性离子转运降低导致细胞内GSH库减少所致。用药理学水平的GSH对Ad 5感染的结膜组织进行粘多糖治疗似乎可以反式刺激粘膜GSH分泌并恢复活性离子转运活性，这表明了一种潜在的有用的眼部感染治疗方案。
Conjunctival epithelial cells of pigmented rabbits secrete reduced glutathione (GSH) into the apical (mucosal) fluid. The aim of the current study was to determine the effect of oxidative stress resulting from viral infection and that of GSH supplementation on redox status, GSH, and ion transport in freshly excised conjunctival tissues and epithelial cell layers in primary culture (RCEC) of adenovirus type 5 (Ad5)-infected rabbits. Lipid peroxidation (LPO) products, nitric oxide (NO), and expression of nitric oxide synthase (NOS2) were quantitated as a function of time after viral inoculation. Unidirectional fluxes of [3H]GSH and changes in short-circuit current (Isc) from mucosal supplementation of Ad5-inoculated conjunctival tissues with GSH and glutathione monoethyl ester (GSH-MEE) were also measured. Ad5 inoculation significantly decreased conjunctival GSH level by 19, 45, 48, and 50% at 8, 24, 48, and 72 h postinfection, respectively. LPO product and NO levels increased significantly (2- and 100-fold, respectively) above that of uninfected controls on Day 3 post-Ad5 inoculation, and co-treatment with GSH-MEE and tocopherol succinate abolished this effect. NO levels showed a progressive increase post-Ad5 inoculation, reaching 0.22 ± 0.06, 8.12 ± 0.91, and 2.05 ± 0.65 μM on Days 1, 3, and 5, respectively, and the highest level was observed on the day of maximal viral replication (Day 3). A very significant induction of the expression of NOS2 on Days 1, 3, and 5 post-Ad5 inoculation was observed. Uninfected control conjunctival tissues displayed a net serosal-to-mucosal GSH flux (Jsm), where the mucosal-to-serosal flux (Jms) was ∼14 pmol h−1cm−2and the Jsmwas ∼22 pmol h−1cm−2. In Ad5-inoculated rabbits similar GSH flux was observed in both the sm and ms directions, and the net GSH flux was negligible. Iscand potential difference (PD) across conjunctival tissues of Ad5-inoculated rabbits decreased by ≥50% compared with control, while the transepithelial electrical resistance (TEER) remained unchanged. Mucosal, but not serosal, superfusion of GSH or GSH-MEE in Ad5-inoculated conjunctival tissues increased the Iscby up to 40% in ∼100 min. Our results show that net secretion of GSH across rabbit conjunctiva is totally blocked after Ad5 inoculation and active ion transport rate decreased by ∼50%. Decreased net GSH secretion into mucosal fluid after Ad5 infection may have resulted from a decreased intracellular GSH pool due to oxyradical-induced changes in redox status and lower active ion transport. Mucosal treatment of Ad5-infected conjunctival tissues with pharmacological levels of GSH appears to transstimulate mucosal GSH secretion and restore active ion transport activity, suggesting a potentially useful therapeutic regimen for ocular infections.