TNF-α下调大鼠肺微血管内皮细胞紧密连接蛋白ZO-1、Claudin-5的表达

TNF-α downregulates the expression of tight junction proteins ZO-1 and Claudin-5 in rat pulmonary microvascular endothelial cells

摘要:

目的 研究肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）对大鼠肺微血管内皮细胞紧密连接蛋白1（zonula occludens-1，ZO-1）、Claudin-5表达的影响，探讨TNF-α对大鼠肺血管屏障的损伤机制。方法 将体外培养的大鼠肺微血管内皮细胞分为对照组、TNF-α 24 h、48 h和72 h组（TNF-α 10 mg/L）。透射电镜观察大鼠肺微血管内皮细胞超微结构。MTT法及流式细胞仪分别测定各组细胞的增殖抑制率及凋亡率。免疫荧光法测定ZO-1及Claudin-5的分布。RT-qPCR及Western blot测定各组细胞ZO-1及Claudin-5的mRNA及蛋白表达。结果 对照组内皮细胞含有大量的线粒体及内质网等结构，结构正常。TNF-α各组细胞线粒体数量减少，线粒体肿胀，嵴溶解，核固缩，凋亡细胞增多，细胞增殖抑制率及凋亡率较对照组增加（均P<0.05），呈时间依赖性。ZO-1及Claudin-5呈线状荧光分布于内皮细胞间连接部位。与对照组比较，TNF-α各组细胞ZO-1的转录及表达水平均降低（均P<0.05）。TNF-α各组Claudin-5的转录水平较对照组明显降低（均P<0.05），TNF-α 48 h、72 h组Claudin-5的蛋白表达水平较对照组降低（均P<0.05），TNF-α 24 h组与对照组比较差异无统计学意义（P>0.05）。结论 TNF-α下调大鼠微血管内皮细胞ZO-1及Claudin-5的表达，诱导细胞损伤及凋亡，破坏肺屏障，导致肺损伤。

Abstract:

Objective To investigate the effect of tumor necrosis factor-α（TNF-α） on the expression of tight junction proteins ZO-1 and Claudin-5 in rat pulmonary microvascular endothelial cells and to explore the injury mechanism of TNF-α on pulmonary vascular barrier in rats.Methods Rat pulmonary microvascular endothelial cells cultured in vitro were divided into control group，and TNF-α 24 h，48 h，and 72 h groups（TNF-α 10 mg/L）. The ultrastructure of rat pulmonary microvascular endothelial cells was observed by a transmission electron microscope. The proliferation inhibition rate and apoptosis rate of cells in each group were determined by MTT assay and flow cytometry. The distribution of ZO-1 and Claudin-5 was determined by the immunofluorescence method. The mRNA and protein expression levels of ZO-1 and Claudin-5 in cells of each group were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot.Results Endothelial cells in the control group contained a large number of mitochondria and endoplasmic reticulum，with a normal structure. Cells in the TNF-α groups showed a decrease in the number of mitochondria，mitochondrial swelling，cristae lysis，karyopyknosis，and an increase in apoptotic cells，with a significant time-dependent increase in the proliferation inhibition rate and apoptosis rate compared with the control group（all P<0.05）. ZO-1 and Claudin-5 were distributed at the junction of endothelial cells，showing linear fluorescence. Compared with the control group，the TNF-α groups showed a significant decrease in the transcription and expression levels of ZO-1 and the transcription level of Claudin-5（P<0.05）； the TNF-α 48 h and 72 h groups showed a significant decrease in the protein expression level of Claudin-5（P<0.05）； the TNF-α 24 h group showed no significant difference（P>0.05）.Conclusion TNF-α downregulates the expression of ZO-1 and Claudin-5 in rat pulmonary microvascular endothelial cells，induces cell injury and apoptosis，destroys the lung barrier，and leads to lung injury.