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

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.