Study On The Mechanism Of High Mobility Group Box 1 Affecting Endothelial Barrier Permeability Through Rho/Rock Pathway

Objective: High mobility group box 1 protein(HMGB1)is an important late inflammatory mediator in the body.In recent years,it has become a research hotspot due to the discovery that it participates in the pathophysiological process of sepsis and plays an important pathogenic role.Sepsis is a very common critical illness in the ICU.The underlying mechanism of its pathogenesis has not been fully elucidated.Tumor necrosis factor(TNF-α),interleukin-1(IL-1),etc.are important early inflammatory mediators in the onset of sepsis.However,the clinical application of TNF-α and IL-1 antagonists has not achieved satisfactory results.Therefore,the purpose of this study is to explore whether it can protect the endothelial barrier by blocking the downstream signaling pathway of the late inflammatory mediator HMGB1.Methods: Human pulmonary microvascular endothelial cells(HPMEC)were cultured in vitro,and the trypsinized HPMEC was subcultured in culture flasks and randomly divided into 9 groups: blank control group(with the same amount of PBS),rh HMGB1 10 min group(600 ng/m L),rh HMGB1 30 min group(600 ng/m L),rh HMGB1 1h group(600 ng/m L),rh HMGB1 6h group(600 ng/m L),rh HMGB1 24 h group(600 ng/m L),rh HMGB1 24h+RAGE inhibitor(FPS-ZM1)group(600 ng/m L rh HMGB1 + FPS-ZM1 0.05μM),rh HMGB1 60min+Rock inhibitor(Y-27632)group(600 ng/m L rh HMGB1 + Y-27632 10μmol/L),rh HMGB1 24 h + Rock inhibitor(Y-27632)group(600 ng/m L rh HMGB1 + Y-27632 10μmol/L).1.After subcultured human lung microvascular endothelial cells,the survival rate of endothelial cells was determined by CCK-8 proliferation experiment.2.Use Transwell method to detect endothelial cell permeability.3.Use Pull down method to detect the activity expression of Rho-GTP induced by HMGB1.4.Detect the protein expressions of Rock,Total Rho A,MLC,p-MLC,ZO-1 and VE-cadherin by Western blot.5.Use immunofluorescence to observe the expression and distribution of F-actin,ZO-1,and VE-cadherin.Results: 1.After treatment with 600 ng/m L rh HMGB1 for 24 h,there was no significant change in endothelial cell viability compared with the blank control group,and the difference was not totally significant(P>0.05).2.The rh HMGB1 applied at concentrations of 100 ng/m L,200 ng/m L,400 ng/m L,and 600 ng/m L stimulated HPMECs for 24 hours.As the concentration of HMGB1 increased,the permeability of the endothelial barrier also increased significantly.The difference was statistically significant(P<0.05)After treating endothelial cells with rh HMGB1(600 ng/m L)for 10 min,30min,and 1h,the permeability of the endothelial cell barrier did not change significantly compared with the blank control group(P>0.05).After treatment with rh HMGB1 for 6 h and 24 h,compared with the blank control group,the permeability of the endothelial cell barrier was significantly increased,and the difference was statistically significant(P<0.05).3.Pull down method and western blotting method showed that HMGB1 induced rapid activation of Rho protein and Rock protein,myosin light chain was activated at 30 min,and its phosphorylation form increased,and the phosphorylation level reached the highest value at 1h,but as time goes by,phosphorylated myosin gradually inactivated.The expression of tight junction protein ZO-1 and adhesion junction protein VE-cadherin gradually decreased after stimulation of rh HMGB1 for 1hour.4.By comparing with the control group,the confocal microscope shows that the rh HMGB1 treatment group can cause changes in the structure and distribution of the HPMECs cytoskeleton protein F-actin in a short period of time.As time goes by,ZO-1loses continuity,distribution breaks,and fluorescence The intensity is weakened.VE-cadherin is destroyed,cross-linked with each other,and distributed in fragments.5.Application of FPS-ZM1 and Y-27632 pretreatment can see its protective effect on HMGB1-mediated endothelial barrier damage.By comparing with the control group,the cell permeability is significantly reduced.Quantitative analysis shows the phosphorylation of MLC.The level is significantly reduced,the protein expression of ZO-1 and VE-cadherin is significantly up-regulated,the stress fibers formed by F-actin aggregation are reduced,the fluorescence expression of ZO-1 and VE-cadherin is obviously increased,and the membrane positioning is significantly increased,which is different from the rh HMGB1 treatment group All were statistically significant(all P<0.05);and Y-27632 was added within the last 4 hours of rh HMGB1 stimulation 24 h,and it was found that it did not protect the endothelial barrier damage.Compared with the rh HMGB1 stimulation group,there is no significant difference in endothelial barrier permeability,and the expression and distribution of ZO-1 and VE-cadherin are still at a low level.Conclusion: After HMGB1 binds to the extracellular RAGE receptor,it induces phosphorylation of MLC,F-actin formation of stress fibers,and changes in the distribution and expression of endothelial connexin through early activation of the Rho/ROCK signaling pathway,leading to contraction of endothelial cells and formation of fissures between cells and increasing the permeability of endothelial cell barrier.