Induction of DDIT4 Impairs Autophagy Through Oxidative Stress in Dry Eye

作者：Bowen Wang, Lulu Peng, Hong Ouyang, Li Wang, Dalian He, Jing Zhong, Yichen Xiao, Yuqing Deng, Meng Li, Saiqun Li, Jin Yuan · 发表于：Investigative Ophthalmology & Visual Science · 年份：2019 · DOI：10.1167/iovs.19-27072 · 被引用次数：59 · 研究领域：Ocular Surface and Contact Lens、Autophagy in Disease and Therapy、Corneal Surgery and Treatments

Purpose: To assess how DNA damage-inducible transcript 4 (DDIT4) and autophagic flux are altered in dry eye disease and reveal the underlying mechanisms. Methods: 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 fun...