State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen University, Guangzhou, Guangdong, China.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen University, Guangzhou, Guangdong, China.
Exp Eye Res. 2021 Apr;205:108491. doi: 10.1016/j.exer.2021.108491. Epub 2021 Feb 12.
This study aimed to investigate the protective effect of melatonin on the corneal epithelium in dry eye disease(DED) and explore its underlying mechanism. Human corneal epithelial(HCE) cells was exposure to t-butylhydroperoxide(tBH), C57BL/6 mice were injected of subcutaneous scopolamine to imitate DED. Melatonin was used both in vivo and in vitro. Cell viability was detected by Cell Counting Kit-8 assay and Lactate Dehydrogenase Leakage. The change of cellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptosis was analyzed by flow cytometry. Western blot assays and immunofluorescence were carried out to measure protein changes. mRNA expression was investigated by RNA sequencing (RNA-Seq) and quantitative real-time PCR. The change of autophagic flux were observed through mCherry-GFP-LC3 transfection and electron microscopy(TEM). Clinical parameters of corneal epithelium defects, conjunctival goblet cells, tear volume, and level of ocular surface inflammation was recorded. Melatonin was able to reduce excessive ROS production and maintain mitochondrial function. TEM assay found melatonin rescued impaired autophagic flux under tBH. Moreover, melatonin significantly preserved cell viability, abolished LDH release, and decreased apoptosis. RNA-Seq indicated that melatonin greatly activating hemeoxygenase-1 (HO-1) expression. Interestingly, HO-1 ablation largely attenuated its protective effects. Besides, in dry eye mouse model, intraperitoneal injection of melatonin showed greatly improved clinical parameters, inhibited activated NLRP3 inflammation cascade, and increased density of goblet cells and tear volume. Thus, melatonin protects corneal epithelial cells from oxidative damage, maintain normal level of autophagy, and reduce inflammation via trigging HO-1 expression in DED.
本研究旨在探讨褪黑素对干燥性角结膜炎（DED）角膜上皮的保护作用，并探讨其潜在机制。我们将人角膜上皮（HCE）细胞暴露于 t-丁基过氧化物（tBH）中，并用皮下注射东莨菪碱的 C57BL/6 小鼠来模拟 DED。褪黑素用于体内和体外实验。通过细胞计数试剂盒-8 检测法和乳酸脱氢酶漏出检测法检测细胞活力。通过流式细胞术分析细胞内活性氧（ROS）水平、线粒体膜电位（MMP）和细胞凋亡的变化。通过 Western blot 检测法和免疫荧光法测量蛋白变化。通过 RNA 测序（RNA-Seq）和实时定量 PCR 检测 mRNA 表达。通过 mCherry-GFP-LC3 转染和电子显微镜（TEM）观察自噬流的变化。记录角膜上皮缺损、结膜杯状细胞、泪液量和眼表炎症水平等临床参数的变化。褪黑素能够减少过多的 ROS 产生并维持线粒体功能。TEM 实验发现褪黑素可挽救 tBH 下受损的自噬流。此外，褪黑素可显著提高细胞活力、消除 LDH 释放和减少细胞凋亡。RNA-Seq 表明褪黑素可大大激活血红素加氧酶-1（HO-1）的表达。有趣的是，HO-1 缺失大大减弱了其保护作用。此外，在干燥性角结膜炎小鼠模型中，腹腔内注射褪黑素可显著改善临床参数，抑制激活的 NLRP3 炎症级联反应，并增加杯状细胞密度和泪液量。因此，褪黑素通过触发 HO-1 表达来保护角膜上皮细胞免受氧化损伤、维持正常的自噬水平，并减少 DED 中的炎症。
