Eye Institute & Affiliated Xiamen Eye Center, School of Medicine, Xiamen University, Xiamen, China.
Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, China.
J Cell Mol Med. 2019 Jun;23(6):4217-4228. doi: 10.1111/jcmm.14310. Epub 2019 Apr 11.
Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure-induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3-methyladenine (3-MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy-related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy-related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress-related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway-related proteins. Real-time quantitative PCR (qRT-PCR) determined IL-1β, IL-6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure-induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress-induced autophagy promoted cell survival. Taken together, air exposure-induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.
不完全的泪膜铺展和眼睑闭合可导致眼表面更新缺陷和空气暴露诱导的上皮角膜病变 (EK)。在这项研究中，我们研究了自噬在介导导致 EK 的眼表面变化中的作用。分别用人角膜上皮细胞 (HCEC) 和 C57BL/6 小鼠作为 EK 模型。透射电子显微镜 (TEM) 评估了空气暴露后 HCEC 的变化。这些模型中的每一个都用自噬抑制剂 [氯喹 (CQ) 或 3-甲基腺嘌呤 (3-MA)] 或激活剂 [雷帕霉素 (Rapa)] 处理。免疫组织化学评估自噬相关蛋白 LC3 和 p62 的表达水平。Western blot 证实自噬相关蛋白 [Beclin1 和哺乳动物雷帕霉素靶蛋白 (mTOR)]、内质网 (ER) 应激相关蛋白 (PERK、eIF2α 和 CHOP) 和 PI3K/Akt/mTOR 信号通路相关蛋白的表达水平。实时定量 PCR (qRT-PCR) 测定 IL-1β、IL-6 和 MMP9 基因表达水平。TUNEL 检测法检测凋亡细胞。TEM 在两种 EK 模型中均发现自噬空泡。增加的 LC3 点状形成和减少的 p62 免疫荧光染色和 Western blot 证实了自噬的诱导。CQ 处理增加了 HCEC 中的 TUNEL 阳性染色，而 Rapa 则有相反的效果。同样，CQ 注射增强了小鼠角膜上皮的空气暴露诱导的凋亡和炎症，而 Rapa 处理则抑制了这种作用。此外，两种 EK 模型中 PERK 和 eIF2α 的磷酸化状态以及 CHOP 表达增加表明 ER 应激诱导的自噬促进了细胞存活。总之，空气暴露诱导的自噬对于维持角膜上皮生理学和细胞存活是必不可少的。
