| Part oneTHE EFFECT OF ULINASTATIN ON THE PERMEABILITY OF VASCULAR ENDOTHELIAL CELLS AND THE MOLECULAR MECHANISM INDUCED BY TNF-Α Objective: To investigate the influence of Ulinastatin(UTI)on the high permeability of vascular endothelial cells induced by tumor necrosis factor alpha(TNF-α)and the molecular mechanism.Methods: 1.TNF-α treated Human umbilical vein endothelial cell line(EA.hy926)was employed as an inflammation model.Horseradish peroxidase(HRP)permeability assay and epithelial voltameter(EVOM)method were performed to measure the permeability of EA.hy926 cells respectively;2.Immunocytochemistry assay was used to observe the expression of p-MYPT1 and the distribution and morphology of F-actin;the expressions of the key molecules related to the vascular endothelial permeability(Rho A,ROCK2,MYPT1,p-MYPT1 and VE-cadherin)were detected by Immunocytochemistry assays,Western blot assays and Quantitative real-time polymerase chain reaction(q RT-PCR)analysis.Results: 1.The transendothelial electrical resistance(TEER)of EA.hy926 cells decreased and the permeability of the cells increased obviously after incubation with TNF-α(P<0.05).The expression of p-MYPT1 was increased and the VE-cadherin was decreased compared with control group,and the F-actin was redistributed,with the formation of additional stress fibers in the cells.While,those phenomena described above could be improved by UTI.2.The results of Immunocytochemistry assays and Western blot assays showed that the expressions of Rho A and ROCK2 were significantly upregulated in the cells treated with TNF-α(P<0.05),however,these two proteins’ high-expression could be inhibited by UTI.Under treatment of TNF-α and UTI,compared with non-tranfected EA.hy926 cells,overexpression of Rho A induced an upregulation of expressions of Rho A,ROCK2 and p-MYPT1,a downregulation of VE-cadherin,as well as an alleviation of high permeability(P<0.05).Conclusions: Ulinastatin could inhibit the hyperpermeability of vascular endothelial cells induced by TNF-α in a dose-dependent manner.And this inhibitory effect of UTI may be related to the Rho/ROCK signaling pathway.Part twoTHE PROTECTIVE EFFECT AND ITS POSSIBLE MECHANISMS OF ULINASTATIN ON THE LUNG INJURY OF THE SEPTIC MICE INDUCED BY LIPOPOLYSACCHARIDE Objective: To investigate the influence of Ulinastatin(UTI)on the lung injury of the septic mice induced by lipopolysaccharide(LPS)and clarify the molecular mechanism.Methods: 1.Septic mice model was induced by intravenous administration of LPS.The C57BL/6 mice were randomly divided into 3 groups: control group(Con group,n=10),model group(LPS group,n=10)and UTI intervention group(L+U group,n=30);2.Lung wet /dry weight(W/D)ratio and Evans Blue(EB)were used to detect the water content and the capillarity permeability respectively;3.HE staining was used to observe the pulmonary histopathology;4.The expressions of VE-cadherin and ROCK2 were measured by immunohistochemical assay.Results: 1.Compared with control group,both the W/D ratio and EB content of lung tissue increased significantly in LPS group(all P<0.05),while in L+U group,the W/D ratio and EB content of lung tissue decreased obviously.When the mice were treated with UTI at the dose of 104 U/kg and 105 U/kg,a significant decrease in both the W/D ratio and EB content of lung were observed compared with LPS group(all P<0.05);2.The destruction of lung tissue in LPS group was serious compared with control group,and the phenomena could be improved by UTI;3.Immunohistochemical results showed that a significant downregulation of VE-cadherin expression and a significant upregulation ROCK2 expression in LPS group compared with control group(all P<0.05),while this phenomena could be alleviated after the treatment with UTI(all P<0.05).Conclusion: UTI could improve the lung injury in a dose-dependent manner by alleviating the vascular hyper-permeability in the septic mice model induced by LPS,while this effect may be related to the Rho/ROCK signaling pathway. |
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