Influence Of Heterogeneous Nuclear Ribonucleoprotein A2/B1 On LPS-induced Vascular Endothelial Cell Injury And Its Mechanisms

Sepsis is a life-threatening organ dysfunction caused by a host response to a pathogen infection.Septic shock,as the most serious form of sepsis,is often accompanied by severe hypotension,microvascular perfusion damage and organ damage,eventually leading to multiple organ failure.Because the mechanism of sepsis is not yet fully clear,sepsis therapy is limited to liquid resuscitation and symptomatic treatment.Vascular endothelial dysfunction is a major factor in sepsis.When sepsis occurs,vascular endothelium is activated,it rapidly becomes a state of pro-coagulation,anti-fibrosis and pro-adhesion.The activation of endothelium is associated with leukocyte aggregation,vascular permeability,inflammatory response and microcirculatory blood flow in sepsis.Thus,the vascular endothelium plays a critical role in sepsis,it could be a potential target for sepsis treatment.In recent years,lots of attention is paid to heterogeneous nuclear ribonucleoproteins A2/B1(hn RNP A2/B1).As a member of heterogeneous nuclear ribonucleoproteins family,hn RNP A2/B1 plays an important role in m RNA splicing,nuclear-cytoplasmic transportation,post-transcriptional regulation and chromosome stability regulation.Also,hn RNP A2/B1 regulates cellular biological functions such as cellular metabolism,migration and invasion,proliferation and mitochondrial stress.It was found that hn RNP A2/B1 could regulate the cell cycle by regulating the transcriptional level of CDKs inhibitor p16INK4 a,P21 and P27.Recently,it has been found that hn RNP A2/B1 is involved in the transformation of lung cancer epithelial cells by modulating E-cadherin expression.The protein expression level of hn RNP A2/B1 was positively correlated with the deletion of E-cadherin in pancreatic tumor tissues.However,the effect of hn RNP A2/B1 in sepsis-induced vascular endothelial injury has not been reported.Our research was to investigate the effect of hn RNP A2/B1 on the pulmonary vascular endothelial injury and its possible mechanisms exposed to LPS.Firstly,we investigated the function of hn RNP A2/B1 on vascular endothelial cells stimulated by LPS.Based on the protection of hn RNP A2/B1 in sepsis-induced endothelial injury,the potential mechanism was further explored.Part 1 The effect of hn RNP A2/B1 on LPS-induced vascularendothelial cells injuryObjectiveTo investigate the effect of silencing hn RNP A2/B1 on LPS-induced vascular endothelial cell injury.MethodsThe effect of silencing hn RNP A2/B1 on LPS-induced HUVECs was studied by silencing hn RNP A2/B1 by si RNA transfection technique.The cells were divided into four groups: hn RNP A2 / B1 si RNA group,negative control(NC)si RNA group,hn RNP A2/B1 si RNA-LPS group and NC si RNA-LPS group,transmembrane electrical resistance and FITC-dextrans were measured.Scratches tests and Transwell migration experiments were detected to evaluated the migration function of endothelium.The changes of endothelial cell adhesion-related molecules were detected by Western blot.Results1.It is showed that the m RNA and protein expression of hn RNP A2/B1 in hn RNP A2/B1 si RNA-664 group were lower than those in NC-si RNA group.The difference was statistically significant(P <0.05).2.The TEER value in NC si RNA-LPS group was lower than that in NC si RNA group.Also,the TEER value in hn RNP A2/B1 si RNA-LPS group was significantly lower than that in NC si RNA-LPS group.The fluorescence intensity of FITC-dextrans in NC si RNA-LPS group was higher than that in NC si RNA group after 12 hours of LPS stimulation.The fluorescence intensity of FITC-dextrans in hn RNPA2/B1 si RNA-LPS group was significantly higher than that in NC si RNA-LPS group(P < 0.05).3.The migration distance of hn RNP A2/B1 si RNA-LPS group was significantly shorter than that in NC si RNA-LPS group.The number of cells in hn RNP A2/B1 si RNA-LPS group was significantly reduced,compare with NC si RNA-LPS group(P<0.05).4.The expression of VE-Cadherin in NC si RNA-LPS group was significantly lower than that in NC si RNA group.The expression of VE-Cadherin in hn RNP A2/B1 si RNA-LPS group was significantly lower than that in NC si RNA group.The expression of ICAM-1 in NC si RNA-LPS group was higher than that in NC si RNA group.The expression of ICAM-1 in hn RNP A2/B1 si RNA-LPS group was significantly higher than that in NC si RNA-LPS group(P <0.05).ConclusionDuring vascular endothelial injury induced by LPS,silencing hn RNP A2/B1 significantly increased the permeability of vascular endothelium,blocked the migration funciton of vascular endothelial cells,up-regulated ICAM-1 and downregulated VE–Cadherin expression,aggravating the destruction of vascular endothelial barrier function.It is suggested that hn RNP A2/B1 may play a protective role in sepsis-induced vascular endothelial injury and it may help to reduce the damage to vascular barrier.Part 2 The potential mechanism of hn RNP A2/B1 in LPS-inducedvascular endothelial cell injuryObjectiveTo investigate the potential mechanism of hn RNP A2/B1 in LPS-induced vascular endothelial cell injury.MethodsThe cells were divided into four groups: hn RNP A2 / B1 si RNA group,negative control(NC)si RNA group,hn RNP A2/B1 si RNA-LPS group and NC si RNA-LPS group.The changes of endothelial cell adhesion-related molecules β-catenin and cdc42 were detected by Western blot after LPS stimulation.ResultsCompared with NC si RNA-LPS group,the expression of β-catenin in hn RNP A2/B1 si RNA-LPS group was lower,while the expression of p-Src in hn RNP A2/B1 si RNA-LPS group was higher(P <0.05).Also,Cdc42 expression levels were not significantly different.ConclusionHn RNP A2/B1 may protect vascular endothelial function in sepsis by up-regulatingβ-catenin and inhibiting Src phosphorylation.