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

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 oc⁃cludens-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-α24 h、48 h和72 h组(TNF-α10 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-α48 h、72 h组Claudin-5的蛋白表达水平较对照组降低(均P<0.05),TNF-α24 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。

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-αon pulmonary vascular barrier in rats.Methods:Rat pulmonary microvascular endothelial cells cultured in vitro were divided into control group,and TNF-α24 h,48 h,and 72 h groups(TNF-α10 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-αgroups showed a decrease in the number of mitochondria,mito⁃chondrial 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.Com⁃pared with the control group,the TNF-αgroups showed a signifi⁃cant decrease in the transcription and expression levels of ZO-1 and the transcription level of Claudin-5(P<0.05);the TNF-α48 h and 72 h groups showed a significant decrease in the protein expres⁃sion level of Claudin-5(P<0.05);the TNF-α24 h group showed no significant difference(P>0.05).Conclusion:TNF-αdownregu⁃lates the expression of ZO-1 and Claudin-5 in rat pulmonary microvascular endothelial cells,induces cell injury and apoptosis,de⁃stroys the lung barrier,and leads to lung injury.