A study of inhibitory effect of ulinastatin on hyper-permeability of endothelium induced by TNF-α

Abstract

Objective To investigate the influence of Ulinastatin(UTI) on the hyper-permeability of vascular endothelial cells induced by tumor necrosis factor alpha (TNF-α). Methods Inflammation model was induced by TNF-α in human umbilical vein endothelial cell line (EA.hy926). The experiment was designed into 4 groups: normal group, TNF-α group, UTI group and TNF-α with UTI(U+ T) group. Methyl thiazolyl tetrazolium (MTT) method and epithelial voltameter (EVOM) method were used to measure cell viability [absorbance (A) value] and transepithelial electrical resistance (TER) of EA.hy926 cells respectively. The expression of VE-cadherin was measured by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. Results Compared with normal group, the TER of EA.hy926 cells induced by TNF- α was significantly decreased (67.200±8.937 vs. 33.600±8.771, P=0.010). The permeability in EA.hy926 cells increased obviously. The hyper- permeability of EA.hy926 cells induced by TNF- α could be alleviated by UTI at the concentrations of 1-100 U/mL in a dose-dependent manner (40.133±7.484 vs. 33.600±8.771, P=0.382; 49.232±3.162 vs. 33.600±8.771, P=0.044; 63.700±8.515 vs. 33.600±8.771, P=0.013). The expression of VE-cadherin mRNA reduced significantly in the TNF-α group (1.089±0.018 vs. 0.835±0.021, P=0.000) compared with normal group. This effect of TNF-α could be attenuated by UTI. When EA.hy926 cells exposed to UTI at 10 U/mL and 100 U/mL, a significant increase of the expression of VE-cadherin mRNA was observed(0.976±0.014 vs. 0.835±0.021, P=0.001; 1.115±0.015 vs. 0.835±0.021, P=0.000). And the inhibition of UTI manifested a dose-dependent manner (1-100 U/mL). The results of the immunocytochemistry showed that the expression of VE-cadherin in TNF-α group was decreased significantly (0.061±0.013 vs. 0.093±0.014, P=0.049) compared with normal group.And the low-expression of VE-cadherin could be alleviated by UTI(0.032±0.004 vs. 0.061±0.013, P=0.016). Conclusion The high permeability of EA.hy926 cells induced by TNF-α could be inhibited by UTI at the concentrations of 1-100 U/mL in a dose-dependent manner. Key words: Human umbilical vein endothelial cell line; High permeability; Ulinastatin; Tumor necrosis factor alpha (TNF-α); VE-cadherin