Induction of DDIT4 Impairs Autophagy Through Oxidative Stress in Dry Eye
Induction of DDIT4 Impairs Autophagy Through Oxidative Stress in Dry Eye
复制标题
干眼症中 DDIT4 的诱导通过氧化应激损害自噬
DOI：
10.1167/iovs.19-27072
复制
发表时间：
2019
影响因子：
4.4
通讯作者：
Yuan Jin
中科院分区：
文献类型：
--
作者：
Wang Bowen;Peng Lulu;Ouyang Hong;Wang Li;He Dalian;Zhong Jing;Xiao Yichen;Deng Yuqing;Li Meng;Li Saiqun;Yuan Jin
目的：评估 DNA 损伤诱导转录物 4 (DDIT4) 和自噬流在干眼病中如何改变，并揭示其潜在机制方法：C57BL/6 小鼠暴露于干燥应激（皮下注射东莨菪碱 [0.5 mg/0.2 mL]，每天 3 次，湿度 < 30%）7 天。原代人角膜上皮细胞和来自人角膜上皮细胞系的细胞在高渗透压下培养。使用蛋白质印迹测定和免疫荧光染色来测量蛋白质表达的变化。通过RT-PCR和定量实时PCR分析mRNA表达。通过电子显微镜观察自噬体。通过 2', 7'-二氯二氢荧光素二乙酸酯和线粒体膜电位测定检测细胞活性氧和线粒体功能。使用 Cell Counting Kit-8 和乳酸脱氢酶测定来测量细胞死亡。采用Annexin V-PI流式细胞术分析细胞凋亡情况。结果：在体外角膜上皮细胞和干眼小鼠模型中观察到微管相关蛋白1轻链3（LC3-II）、隔离体1（SQSTM1）和DDIT4表达增加。电子显微镜显示，在高渗透压下，人角膜上皮细胞中存在较大的自噬空泡，其材料降解不良。此外，我们发现DDIT4敲低通过破坏活性氧释放并恢复高渗透压下的线粒体功能，显着抑制LC3-II和SQSTM1的表达。此外，DDIT4的消除有效地保留了细胞活力并抑制了细胞凋亡。结论：通过DDIT4诱导释放过量的活性氧可导致干眼病中自噬受损和细胞活力下降。
Purpose: To assess how DNA damage-inducible transcript 4 (DDIT4) and autophagic flux are altered in dry eye disease and reveal the underlying mechanismsMethods: C57BL/6 mice were exposed to desiccating stress (subcutaneous scopolamine [0.5 mg/0.2 mL] 3 times a day, humidity< 30%) for 7 days. Primary human corneal epithelial cells and cells from a human corneal epithelial cell line were cultured under hyperosmolarity. Western blot assays and immunofluorescence staining were used to measure changes in protein expression. mRNA expression was analyzed by RT-PCR and quantitative real-time PCR. Autophagosomes were observed through electron microscopy. Cellular reactive oxygen species and mitochondrial function were detected with 2′, 7′-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential assays. Cell Counting Kit-8 and lactate dehydrogenase assays were used to measure cell death. Apoptosis was analyzed by Annexin V-PI flow cytometry.Results: Increased expression of microtubule-associated protein 1 light chain 3 (LC3-II), sequestosome 1 (SQSTM1), and DDIT4 were observed in corneal epithelial cells in in vitro and mice models of dry eye. The electron microscopy revealed large autophagic vacuoles with poorly degraded materials in human corneal epithelial cells under hyperosmolarity. In addition, we found that DDIT4 knockdown significantly suppressed the expression of LC3-II and SQSTM1 by disrupting reactive oxygen species release and restoring mitochondrial function under hyperosmolarity. Moreover, the ablation of DDIT4 effectively preserved cell viability and inhibited apoptosis.Conclusions: Excessive reactive oxygen species release through DDIT4 induction can lead to impaired autophagy and decreased cell viability in dry eye disease.