Zhang Yun, Yang Ming, Zhao Shi-Xin, Nie Li, Shen Li-Jun, Han Wei
Eye Centre, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, Zhejiang Province, China.
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, Zhejiang Province, China.
Int J Ophthalmol. 2022 May 18;15(5):683-689. doi: 10.18240/ijo.2022.05.01. eCollection 2022.
To investigate the mechanism of the tight junction (TJ) disruption and the association between tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMPs) under hyperosmotic condition in primary human corneal epithelial cells (HCECs).
The cultured HCECs were exposed to media which adding sodium chloride (NaCl) for hyperosmolar stress or adding rh-TNF-α (10 ng/mL). NF-κB inhibitor (5 µmol/L) or GM-6001 (potent and broad spectrum MMP inhibitor, 20 µmol/L) was added 1h before that treatment. The integrity of TJ proteins was determined by immunofluorescent (IF) staining. The mRNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the protein expression by enzyme-linked immunosorbent assay (ELISA).
TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The mRNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 mOsM. TNF-α mediated the expression and production of MMP-1, MMP-13, MMP-9, and MMP-3 stimulated by hyperosmotic stress. The production of MMPs in hyperosmolar media were increased through the increase of TNF-α. GM-6001 prevent the destruction of ZO-1 and Occludin in hyperosmolar stress and rh-TNF-α treated medium. TNF-α induced activation of MMPs was involved in the TJ disruption by hyperosmolarity.
TJ proteins ZO-1 and Occludin are disrupted by hyperosmolar stress and TNF-α, but protected by MMP inhibitor (GM-6001). It suggests that TNF-α/MMP pathway mediates the TJ disruption in primary HCECs exposed to hyperosmotic stress.
研究高渗条件下原代人角膜上皮细胞（HCECs）中紧密连接（TJ）破坏的机制以及肿瘤坏死因子（TNF）-α与基质金属蛋白酶（MMPs）之间的关联。
将培养的HCECs暴露于添加氯化钠（NaCl）以产生高渗应激的培养基中，或添加重组人TNF-α（10 ng/mL）。在该处理前1小时添加NF-κB抑制剂（5 μmol/L）或GM-6001（强效广谱MMP抑制剂，20 μmol/L）。通过免疫荧光（IF）染色确定TJ蛋白的完整性。通过定量逆转录聚合酶链反应（RT-qPCR）评估TNF-α和MMPs的mRNA水平，并通过酶联免疫吸附测定（ELISA）评估蛋白表达。
暴露于高渗培养基的原代HCECs中TJ蛋白ZO-1和闭合蛋白被破坏。在500 mOsM的高渗培养基中，TNF-α的mRNA表达和蛋白产生显著增加。TNF-α介导高渗应激刺激的MMP-1、MMP-13、MMP-9和MMP-3的表达和产生。高渗培养基中MMPs的产生通过TNF-α的增加而增加。GM-6001可防止高渗应激和rh-TNF-α处理的培养基中ZO-1和闭合蛋白的破坏。TNF-α诱导的MMPs激活参与了高渗引起的TJ破坏。
TJ蛋白ZO-1和闭合蛋白被高渗应激和TNF-α破坏，但受MMP抑制剂（GM-6001）保护。这表明TNF-α/MMP途径介导了暴露于高渗应激的原代HCECs中的TJ破坏。
