The Role And Mechanism Of Ezrin Protein On Hyperpermeability Of Rat Pulmonary Microvascular Endothelial Cells Induced By TNF-?

BackgroundAcute respiratory distress syndrome(ARDS)can be aggravated by many factors inside and outside the lung.Although continuous efforts have been made to study the drug and non-drug treatment of ARDS,its morbidity and mortality remain high.The high mortality and the lack of effective treatment of ARDS make it a thorny problem that perplexes the clinic,especially in the Intensive Care Unit.The pathogenesis of ARDS is complex and unknown,involving different immune cells from innate and acquired immune systems to produce different inflammatory immune cytokines,resulting in increasing pulmonary microvascular permeability,and inducing non-cardiogenic pulmonary edema,causing respiratory failure and respiratory distress in patients.However,the inflammatory immune net is complex,so it is difficult to find an exact inflammatory factor as a molecular therapy target.Therefore,we imagine whether we can explore the pathogenesis of ARDS from the point of view of increasing the permeability of pulmonary microvascular endothelial cells(PMVECs)in ARDS,and look forward to finding molecular targets for the treatment of ARDS from the downstream of complex inflammation and immune response.Purpose1.Observe the distribution and expression level of Ezrin protein in PMVECs hyperpermeability induced by inflammatory immune factor tumor necrosis factor-?(TNF-?).2.Explore whether Ezrin protein participates in the increase of PMVECs permeability induced by TNF-?through Ras homolog gene family,member A(RhoA)or focal adhesion kinase(FAK)signalling pathway.3.Detect the relationship between RhoA signal pathway and FAK signalling pathway in the increase of PMVECs permeability induced by TNF-?.Method1.Rat PMVECs were isolated and cultured.The morphology of rat PMVECs was observed under inverted microscope and Fluorescein isothiocyanate phytohemagglutinin(FITC-BSI)was used to identify rat PMVECs.2.Rat PMVECs was seeded into Transwell chamber and cultured to construct rat PMVECs monolayer model in vitro.3.Observe the response of rat PMVECs monolayer to TNF-?stimulation.3.1 The change of transendothelial electrical resistance(TER)was observed in rat PMVECs after TNF-?stimulation.3.2 The expression level of phosphorylated Ezrin/Ezrin protein were analyzed in rat PMVECs treated with TNF-?.4.The role of Ezrin short hairpin RNA(shRNA)in PMVECs endothelial response induced by TNF-?.4.1 The effect of Ezrin shRNA on the distribution of phosphorylated Ezrin/Ezrin protein were observed by confocal laser scanning microscope and flow cytometry in TNF-?-induced PMVECs.4.2 The effect of Ezrin shRNA on the expression level of phosphorylated Ezrin/Ezrin protein was detected by western blotting in TNF-?-induced PMVECs.4.3 Observe the effect of Ezrin shRNA on endothelial permeability of PMVECs induced by TNF-?(methods:TER and fluorescein isothiocyanate-bovine serum albumin(FITC-BSA)).5.The role of Ezrin protein threonine 567site mutant(Ezrin^T567A)in the endothelial response of PMVECs induced by TNF-?.5.1 Observe the effect of Ezrin^T567A on the distribution of phosphorylated Ezrin/Ezrin protein in TNF-?-induced PMVECs.5.2 Detect the effect of Ezrin^T567A on the expression level of phosphorylated Ezrin/Ezrin protein in TNF-?-induced PMVECs.5.3 Observe the effect of Ezrin^T567A on endothelial permeability of PMVECs induced by TNF-?.6.The role of RhoA signalling pathway in TNF-?-induced Ezrin protein phosphorylation and endothelial permeability.6.1 The changes of Ezrin protein phosphorylation and endothelial permeability were observed via RhoA inhibitor in TNF-?-induced PMVECs.6.2 The RhoA shRNA vector was constructed and transfected into rat PMVECs.The changes of Ezrin protein phosphorylation and endothelial permeability were observed by RhoA shRNA in TNF-?-induced PMVECs.7.The role of FAK signalling pathway in TNF-?-induced Ezrin protein phosphorylation and endothelial permeability.7.1 The changes of Ezrin protein phosphorylation and endothelial permeability induced were observed by FAK inhibitor in TNF-?-induced PMVECs.7.2 The FAK shRNA vector was constructed and transfected into rat PMVECs.The changes of Ezrin protein phosphorylation and endothelial permeability were observed by FAK shRNA in TNF-?-induced PMVECs.8.Observe the relationship between RhoA signal pathway and FAK signal pathway.8.1 After RhoA shRNA treatment,the change of FAK expression level was observed in TNF-?-induced PMVECs.8.2 After FAK shRNA treatment,the change of RhoA expression level was observed in TNF-?-induced PMVECs.Results1.The rat PMVECs showed the shape of paving stone under the inverted microscope,and the green fluorescence was observed under the inverted fluorescence microscope after FITC-BSI treatment.2.TNF-?induced TER and Ezrin protein phosphorylation in a time-and dose-dependent manner in rat PMVECsThe TER of PMVECs was decreased in a time-and dose-dependent manner after stimulation of TNF-?at different time points(0,0.25,0.5,1,2,3,6,12 h)and at different concentrations(0,0.2,2,20,200 ng/ml).The phosphorylated Ezrin protein was increased in a time-and dose-dependent manner after TNF-?stimulation.However,the expression level of Ezrin protein was not affected by the stimulation of TNF-?.3.Ezrin protein knockdown partially reverses endothelial response induced by TNF-?.Western blotting showed that Ezrin shRNA could effectively knock down the expression level of Ezrin protein.Stimulation with 20 ng/ml of TNF-?for 2 h resulted in the localization of phosphorylated Ezrin protein to the cell periphery and the recombination of filamentous actin(F-actin),resulting in an increase in the expression level of phosphorylated Ezrin protein.The rat PMVECs permeability was significantly increased treated with TNF-?.Ezrin shRNA can partially reverse the localization of phosphorylated Ezrin protein to the cell periphery and F-actin recombination,reduce the expression level of phosphorylated Ezrin protein and alleviate endothelial hyperpermeability induced by TNF-?.The distribution and expression level of Ezrin protein itself did not respond to TNF-?.4.Ezrin^T567A inhibits endothelial response induced by TNF-?.After Ezrin^T567A treatment,there was no change in the distribution and expression level of phosphorylated Ezrin protein in TNF-?-induced PMVECs,indicating that the silencing effect of phosphorylated Ezrin protein was achieved.PMVECs permeability increased significantly after TNF-?stimulation,and decreased significantly after Ezrin T567A transfection.5.TNF-?regulates Ezrin protein phosphorylation and endothelial permeability via RhoA signalling pathway.The effect of RhoA was detected by RhoA inhibitor C3 transferase and RhoA shRNA.After pre-incubated with C3 transferase(2?g/ml)for 6 h,and then stimulated with TNF-?for 2 h,it was found that the peripheral localization of phosphorylated Ezrin protein and the recombination of F-actin were significantly reversed than those in TNF-?group,and the phosphorylation level of Ezrin protein and endothelial permeability were also decreased.After RhoA shRNA treatment,it was found that RhoA shRNA+TNF-?group could also significantly inhibit the change of Ezrin protein phosphorylation induced by TNF-?and protect the endothelial barrier.6.TNF-?regulates Ezrin protein phosphorylation and endothelial permeability via FAK signalling pathway.The role of FAK in endothelial response induced by TNF-?was also tested by FAK inhibitor PF-573228 and FAK shRNA.1?M PF-573228 was given for 0.5 h,and then TNF-?treated for 2 h,it was found that PF-573228+TNF-?group could partially reverse the distribution and expression level of phosphorylated Ezrin protein and reduce the endothelial hyperpermeability induced by TNF-?.7.FAK is the upstream signalling pathway of RhoA in TNF-?-induced endothelial response.After TNF-?stimulation,phosphorylated FAK(p-FAK)and activated RhoA (RhoA-GTP)increased significantly,but the level of FAK and RhoA did not change,indicating that TNF-?could induce the activation of FAK and RhoA.After pretreatment with RhoA shRNA and then stimulated by TNF-?,the level of p-FAK did not change compared with the group stimulated by TNF-?alone,indicating that RhoA shRNA could not inhibit the activation of FAK induced by TNF-?.On the contrary,the RhoA-GTP level of PMVECs in FAK shRNA plus TNF-?group was significantly lower than that of single TNF-?stimulation group,which was similar to the baseline level(p>0.05).It shows that FAK is the upstream signal of RhoA.Conclusion1.TNF-?can induce Ezrin protein phosphorylation and permeability increase in rat PMVECs.2.Phosphorylated Ezrin protein is involved in the regulation of endothelial hyperpermeability in TNF-?-stimulated rat PMVECs.3.TNF-?induces Ezrin protein phosphorylation and endothelial hyperpermeability via RhoA or FAK signalling pathway in PMVECs.4.FAK is the upstream signalling pathway of RhoA in the phosphorylation of Ezrin protein and the increase of endothelial permeability in TNF-?-stimulated rat PMVECs.