Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, P.R. China.
Curr Eye Res. 2024 Jan;49(1):25-32. doi: 10.1080/02713683.2023.2262169. Epub 2024 Jan 3.
To confirm the expression and investigate the role of LC3-associated phagocytosis (LAP) in dry eye disease (DED).
The DED model of mice was established by scopolamine subcutaneous injection in a low-humidity environment chamber. Tear secretion test and corneal fluorescein sodium staining were used to evaluate the severity of DED. Expression levels of Rubicon, microtubule-associated protein light chain 3-II (LC3-II), Beclin-1 and autophagy-related gene-7 (Atg-7) in corneas of mice with DED were tested by western blot. Cell Counting Kit-8 (CCK-8) assay was used to detect the effects of different concentrations of hypertonic solutions on the proliferation activity of human corneal epithelial cells (HCECs). The expression levels of Dectin-1, IL-6 and IL-1β in HCECs after stimulation with different concentrations of hypertonic solutions were tested. The expressions of Rubicon, LC3-II, Beclin-1 and ATG-7 in HCECs were detected by reverse transcription polymerase chain reaction (RT-PCR). After being pretreated with 10 μM si-Rubicon, the severity of the disease was documented by corneal fluorescein sodium staining. And the expression levels of IL-6 and IL-1β were also tested by RT-PCR.
Compared with the normal control group, the corneal fluorescein sodium staining scores and tear secretion were significantly reduced. Rubicon, LC3-II, Beclin-1 and ATG-7 were significantly elevated. CCK-8 showed that the 400 and 450 mOsM hypertonic solutions did not affect the proliferation activity of HCECs. The expression of Dectin-1, IL-1β and IL-6 were elevated after stimulation with 450 mOsM solution. LC3-II, Rubicon, ATG-7 and Beclin-1 increased after stimulation with 450 mOsM hyperosmolar solution in HCECs. Corneal fluorescein staining showed that si-Rubicon increased the severity of DED in mice. Moreover, the mRNA expressions of inflammatory factors IL-1β and IL-6 in the cornea of mice were significantly increased.
DED increased the expression of proteins associated with LAP. LAP could play an anti-inflammatory effect in DED.
证实 LC3 相关噬菌作用（LAP）在干燥性眼病（DED）中的表达及作用。
通过在低湿度环境室中皮下注射东莨菪碱建立 DED 小鼠模型。通过泪液分泌试验和角膜荧光素钠染色评估 DED 的严重程度。通过 Western blot 检测 DED 小鼠角膜中Rubicon、微管相关蛋白轻链 3-II（LC3-II）、Beclin-1 和自噬相关基因-7（Atg-7）的表达水平。用细胞计数试剂盒-8（CCK-8）检测不同浓度高渗溶液对人角膜上皮细胞（HCEC）增殖活性的影响。用不同浓度高渗溶液刺激 HCEC 后，检测 Dectin-1、IL-6 和 IL-1β的表达水平。用逆转录聚合酶链反应（RT-PCR）检测 HCEC 中 Rubicon、LC3-II、Beclin-1 和 ATG-7 的表达。用 10 μM si-Rubicon 预处理后，通过角膜荧光素钠染色记录疾病严重程度，并通过 RT-PCR 检测 IL-6 和 IL-1β的表达水平。
与正常对照组相比，角膜荧光素钠染色评分和泪液分泌明显减少，Rubicon、LC3-II、Beclin-1 和 ATG-7 明显升高。CCK-8 显示 400 和 450 mOsM 高渗溶液不影响 HCEC 的增殖活性。用 450 mOsM 溶液刺激后，Dectin-1、IL-1β 和 IL-6 的表达升高。用 450 mOsM 高渗溶液刺激 HCEC 后，LC3-II、Rubicon、ATG-7 和 Beclin-1 增加。角膜荧光素染色显示，si-Rubicon 增加了 DED 小鼠的疾病严重程度，而且小鼠角膜中炎症因子 IL-1β 和 IL-6 的 mRNA 表达明显增加。
DED 增加了与 LAP 相关的蛋白表达，LAP 在 DED 中可能发挥抗炎作用。
