Zhang Runze, Liang Lihong, Liao Kai, Zeng Hao, Yang Xue, Wang Xiaoran, 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.
Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology and Visual Sciences, Beijing, China.
Invest Ophthalmol Vis Sci. 2025 May 1;66(5):23. doi: 10.1167/iovs.66.5.23.
To reveal the function of autophagy impairment-derived sequestosome 1 (SQSTM1) in inducing ferroptosis in an experimental dry eye model and investigate the underlying mechanism.
To induce the dry eye animal model, 8-week-old C57BL/6 mice were subcutaneously injected with scopolamine and exposed to a desiccated environment. To build the in vitro dry eye model, human corneal epithelial cells (HCECs) were applied with desiccating stress. Cell viability was examined using a CCK-8 kit. Intracellular reactive oxygen species (ROS), oxidative lipid, and Fe2+ were detected using the H2DCFDA assay kit, C11 BODIPY probe, and FerroOrange probe. Gene expression was screened by RNA sequencing. Protein expression was evaluated by western blot and immunofluorescence staining. Corneal defect area was assessed by fluorescein sodium staining. Conjunctiva goblet cells were counted by periodic acid-Schiff staining. Tear secretion was measured using phenol red cotton thread.
Desiccating stress induced ferroptosis and SQSTM1 accumulation in both HCECs and C57BL/6 mice. SQSTM1 knockdown alleviated ferroptosis in HCECs. In contrast, the overexpression of SQSTM1 promoted ferroptotic changes. Additionally, overexpression of SQSTM1 significantly increased acyl-CoA synthetase long chain family member 4 (ACSL4). Also, targeted inhibition of ACSL4 mitigated the dry eye symptoms and ferroptosis caused by both SQSTM1 overexpression and desiccating stress.
The accumulation of SQSTM1 triggers corneal epithelial cells ferroptosis through ACSL4 in dry eye disease.
揭示自噬损伤衍生的聚集体结合蛋白1（SQSTM1）在实验性干眼模型中诱导铁死亡的作用，并探讨其潜在机制。
为诱导干眼动物模型，对8周龄C57BL/6小鼠皮下注射东莨菪碱并置于干燥环境中。为构建体外干眼模型，对人角膜上皮细胞（HCECs）施加干燥应激。使用CCK-8试剂盒检测细胞活力。使用H2DCFDA检测试剂盒、C11 BODIPY探针和FerroOrange探针检测细胞内活性氧（ROS）、氧化脂质和Fe2+。通过RNA测序筛选基因表达。通过蛋白质免疫印迹和免疫荧光染色评估蛋白质表达。通过荧光素钠染色评估角膜缺损面积。通过过碘酸希夫染色计数结膜杯状细胞。使用酚红棉线测量泪液分泌。
干燥应激在HCECs和C57BL/6小鼠中均诱导铁死亡和SQSTM1积累。敲低SQSTM1可减轻HCECs中的铁死亡。相反，SQSTM1的过表达促进了铁死亡变化。此外，SQSTM1的过表达显著增加了酰基辅酶A合成酶长链家族成员4（ACSL4）。此外，靶向抑制ACSL4可减轻由SQSTM1过表达和干燥应激引起的干眼症状和铁死亡。
在干眼病中，SQSTM1的积累通过ACSL4触发角膜上皮细胞铁死亡。
