TNF-α downregulates the expression of tight junction proteins ZO-1 and Claudin-5 in rat pulmonary microvascular endothelial cells
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-α24 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,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-α groups 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-α 48 h and 72 h groups showed a significant decrease in the protein expres-sion level of Claudin-5(P＜0.05);the TNF-α 24 h group showed no significant difference(P＞0.05).Conclusion:TNF-α downregu-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.
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