Rac1 Regulates TNF-α-induced High Permeability Of Rat Pulmonary Microvascular Endothelial Cells Through IQGAP1 And Ezrin Pathways

Background and ObjectiveAcute respiratory distress syndrome(ARDS) is caused by a variety of infectious and aseptic causes,such as pneumonia,inhalation of gastric contents,sepsis,acute liver failure,and acute pancreatitis.The disease,its occurrence and development are complicated,and the pathogenesis has not been fully elucidated.The underlying pathogenesis may be due to neutrophil accumulation and pro-inflammatory cytokines acting on pulmonary microvascular endothelial cells(PMVECs),leading to disruption of intercellular connections(such as adhesion junctions),microtubule activation,and actin Cytoskeleton remodeling,cell contraction and gap formation,the normal pulmonary microvascular permeability barrier is destroyed,protein-rich fluid enters the interstitial lung and alveolar cavity,and clinically severe hypoxic respiratory failure occurs.Studies have shown that a single block of inflammatory mediators has not achieved ideal results.Exploring the signaling pathways related to inflammatory factors to block the occurrence of inflammatory storms is a new hot spot that is currently concerned.Rac1,as a member of the small G protein family,plays an important role in stabilizing the cytoskeleton of actin and maintaining the function of the microvascular barrier.Rac1 regulates the cytoskeleton and intercellular connections by promoting the formation of actin fiber bundles in the cortex and remodeling of the junction complex.There is evidence that inhibiting Rac1 can significantly increase venous microvascular permeability.Membrane-F-actin connexin Ezrin and scaffold protein IQGAP1 are co-localized in the subcellular membrane cytoskeleton,and are the hubs for theaggregation of different signal complexes.In recent years,people have also begun to study their role in endothelial permeability,but how they affect the cell barrier under inflammatory conditions has not been reported.F-actin-mediated adhesion connection is a key factor in maintaining the endothelial barrier.Cortactin promotes its relationship with the cortex actin cytoskeleton by increasing its distribution in the cell cortex.Binding,thereby enhancing adhesion and tight junctions,regulating the dynamics of the cytoskeleton,and participating in the regulation of endothelial barrier function.TNF-α is the main pro-inflammatory cytokine secreted by ARDS in the early stages of the disease.It disrupts the functional integrity of the endothelial cell barrier by altering the tightly connected structure of the endothelial cells and the distribution of F-actin.This study intends to stimulate PMVEC by TNF-α,destroy the cell barrier,and inhibit the expression of Rac1,IQGAP1,and Ezrin,respectively,and observe their role in PMVEC injury induced by TNF-α.Methods1.Isolate,culture,and identify primary RPMVECs in vitro.2.Use transwell chambers to separately construct in vitro RPMVECs monolayer cell models.The transendothelial electrical resistance(TEER)changes and the permeability changes of FITC-labeled BSA(FITC-BSA)were measured after different stimuli were administered according to the corresponding experimental groups.3.Observe the changes of F-actin and cortactin of RPMVECs after stimulation with different drugs with an inverted fluorescence microscope.4.The expression levels of Rac1,IQGAP1,Ezrin,and cortactin in different experimental groups were detected by western blot.5.Construct a targeting vector plasmid and package the corresponding lentivirusinfected RPMVECs,and down-regulate the expression of Rac1,IQGAP1,and Ezrin,respectively.6.After detecting down-regulating the expression of Rac1,IQGAP1,and Ezrin,the changes of TEER,FITC-BSA and cytoskeleton were detected.Results1.The RPMVECs were successfully isolated and cultured in vitro and confirmed by morphology and FITC-phytohemagglutinin binding test.2.CCK8 found that when the TNF-α concentration was 0,1,10,and 100 ng / m L,it had no significant effect on PMVECs cell viability.The TEER on Transwell showed a dose-dependent(1-100 ng / m L)and time-dependent(0-6 h)decrease.FITC-BSA permeability after 2 hours of TNF-α stimulation also increased with the increase of TNF-α concentration.3.The primary RPMVECs were infected with lentivirus 48 hours after the expression of green fluorescent protein(GFP)was visible under a fluorescence microscope,and the expression intensity of GFP was further increased after 72 hours.The infection efficiency,that is,the proportion of the initial value at the end of 72 hours:Rac1-sh RNA 15 ± 7%,IQGAP1-sh RNA 10 ± 1.1%,Ezrin-sh RNA 35 ± 3.16%,and the three proteins in the negative control group had no significant changes.4.After TNF-α(100 ng / m L)stimulated PMVECs,the activity of Rac1(Rac1-GTP)decreased significantly in a time-dependent manner(0-2 h),but the total expression level of Rac1 remained unchanged.Rac1 was down-regulated,and the TEER of the cells was reduced to about 57% of the control group.After 2 hours of stimulation with TNF-α,the TEER of the sh Rac1 group decreased to 36%,while the TEER of the control-sh RNA group decreased to 61%.In contrast,if pre-incubation with8-O-Me-c AMP(200 mmo L / L),TEER in the TNF-α group increased to 130%,which was still slightly higher than the control group at the end of 3 hours.FITC-BSA permeability also shows similar changes in permeability.5.After TNF-α acts on the cells,F-actin aggregates and the distribution of cortactin on the periphery of the cells is reduced.Regardless of whether or not exposedto TNF-α,after the expression of Rac1 was inhibited,high stress fibers appeared in PMVEC cells,and the level of cortactin on the cell surface decreased significantly.PMVECs pretreated with Rac1-specific agonist O-Me-c AMP can reverse the cytoskeleton rearrangement induced by TNF-α.6.After 100 ng / m L TNF-α stimulated PMVECs,the expression level of IQGAP1 decreased in a time-dependent manner(0-3 h).Down-regulation of IQGAP1 reduced the TEER of the cells to 78% of the negative control group.After 2 hours of TNF-αstimulation,the TEER in the IQGAP1-sh RNA group(TNF-α + IQGAP1-sh RNA group)decreased to 34%,while the TEER in the negative control group(TNF-α + controlsh RNA)decreased to 66%.FITC-BSA permeability also shows similar changes in permeability.7.When TNF-α or sh IQGAP1 alone acts on cells,F-actin aggregates,and peripheral cortactin distribution decreases.However,the co-treatment of the two reduced the formation of high-level stress fibers and cortactin distribution in the membrane.Western Blot found that in cells transfected with sh IQGAP1,peripheral cortactin expression was reduced.When exposed to TNF-α,the cortactin content on the membrane was further reduced.8.Downregulating IQGAP1,the expression of Rac1-GTP in the cells was significantly reduced.After 2 hours of TNF-α stimulation,compared with the control group(control-sh RNA + TNF-α group),the cells down-regulated in IQGAP1(sh IQGAP1 + TNF-α group)The reduction of Rac1-GTP is more obvious.9.O-Me-c AMP treatment of sh IQGAP1 transfected cells can reverse the increase in TEER and decrease in FITC-BSA induction induced by TNF-α,reduce cytoskeleton remodeling and intracellular redistribution of cortactin10.After TNF-α stimulation,the active Ezrin(p-Ezrin)increased in a time-dependent manner,but the total intracellular Ezrin expression level remained unchanged.In the resting state,the TEER value of the sh Ezrin group was slightly lowerthan that of the negative control group.Down-regulating Ezrin could restore the TEER reduction caused by TNF-α.The Rac1-specific inhibitor NSC23766 further reduced the TEER reduction induced by TNF-α and sh Ezrin.sh Ezrin can resist the increase of FITC-BSA permeability caused by TNF-α.After adding NSC23766,the FITC-BSA permeability increases again.11.Downregulating Ezrin can significantly reduce F-actin polymerization,stress fiber formation,and Cortactin redistribution in TNF-α-induced RPMVECs,while NSC23766 can inhibit sh Ezrin’s maintenance of TNF-α-induced PMVEC cytoskeleton and cytoskeleton remodeling.Conclusions1.TNF-α can cause PMVEC permeability to increase without affecting cell viability.2.Active Rac1 plays an important role in stabilizing the cytoskeleton and maintaining cell permeability.3.When PMVECs were treated with TNF-α,the expression of IQGAP1 decreased,resulting in a decrease in active Rac1 content,redistribution of F-actin and cortactin,resulting in high cell permeability.4.When PMVECs were treated with TNF-α,the expression of phosphorylated Ezrin increased,resulting in a decrease in active Rac1 content,redistribution of F-actin and cortactin,resulting in high cell permeability.