Zuo Xin, Zeng Hao, Yang Xue, He Dalian, Wang Bowen, Yuan Jin
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2024 Dec 2;65(14):12. doi: 10.1167/iovs.65.14.12.
Ferroptosis occurred in corneal epithelial cells has been implicated in the inflammation in dry eye disease (DED). Given the proposed link between ferroptosis and autophagy, this study aims to investigate the role of autophagy in driving ferroptosis in corneal epithelial cell and enrich the pathogenesis underlying DED.
DED models were established in C57BL/6 mice via scopolamine injection and in human corneal epithelial cell line (HCEC) using hyperosmotic medium. Lipidomic and transcriptomic analysis were conducted to assess lipid metabolism and regulatory pathways. Atg5 expression was manipulated in vivo using cholesterol-modified small interfering RNA. Lipid droplets (LDs) and lysosomes were labeled with BODIPY 493/503 and Lysotracker Red DND-99, respectively. Western blot, immunofluorescence (IF) staining, co-immunoprecipitation (CO-IP), transmission electron microscopy and microplate reader were used to explore protein expressions and interactions, cellular structures, and free fatty acid (FFA) content.
Our results revealed that autophagy was activated in DED, as evidenced by lipidomic and transcriptomic analyses. Enhanced lipophagy was observed in HCECs exposed to hyperosmolarity, manifested by lysosome-LD co-localization and autophagic vacuoles containing LDs. Upregulation of Atg5 promoted lipophagy, leading to elevated cellular FFA levels, lipid peroxidation, and expression of ferroptosis markers. Interaction between Atg5 and perilipin3 was confirmed through CO-IP and IF. In the DED mouse model, Atg5 inhibition effectively ameliorated corneal damage, suppressed ferroptosis and ocular surface inflammation.
Our findings highlight the pivotal role of Atg5-mediated lipophagy in driving ferroptosis in corneal epithelial cells in DED, proposing Atg5 as a promising therapeutic target for mitigating ferroptosis-induced cell damage and inflammation in DED.
角膜上皮细胞发生的铁死亡与干眼病（DED）的炎症有关。鉴于铁死亡与自噬之间的潜在联系，本研究旨在探讨自噬在驱动角膜上皮细胞铁死亡中的作用，并丰富DED的发病机制。
通过注射东莨菪碱在C57BL/6小鼠中建立DED模型，并使用高渗培养基在人角膜上皮细胞系（HCEC）中建立模型。进行脂质组学和转录组学分析以评估脂质代谢和调控途径。使用胆固醇修饰的小干扰RNA在体内操纵Atg5的表达。分别用BODIPY 493/503和Lysotracker Red DND-99标记脂滴（LDs）和溶酶体。采用蛋白质印迹法、免疫荧光（IF）染色、免疫共沉淀（CO-IP）、透射电子显微镜和酶标仪来探索蛋白质表达和相互作用、细胞结构以及游离脂肪酸（FFA）含量。
我们的结果表明，脂质组学和转录组学分析证明DED中自噬被激活。在暴露于高渗环境的HCEC中观察到脂自噬增强，表现为溶酶体与LD的共定位以及含有LD的自噬泡。Atg5的上调促进了脂自噬，导致细胞FFA水平升高、脂质过氧化以及铁死亡标志物的表达。通过CO-IP和IF证实了Atg5与脂滴包被蛋白3之间的相互作用。在DED小鼠模型中，抑制Atg5可有效改善角膜损伤，抑制铁死亡和眼表炎症。
我们的研究结果突出了Atg5介导的脂自噬在驱动DED角膜上皮细胞铁死亡中的关键作用，提出Atg5作为减轻DED中铁死亡诱导的细胞损伤和炎症的有前景的治疗靶点。
