Role Of CREB In The Injury Of Rat Pulmonary Microvascular Endothelial Cell Induced By LPS

AimcAMP response element binding protein (CREB) is an important nuclear transcriptionfactor with complex biological functions, which can regulate transcription of targetgenes containing CRE sequence in promoter. Numerous studies show that CREB mayplay an important role in diseases which vascular barrier damaged, such as acuterespiratory distress syndrome, pulmonary hypertension, diabetic retinopathy andpolycystic ovary syndrome. It is closely related to CREB regulation of vascularpermeability and inflammation. However, the impact of CREB on vascular endothelialcells is complex, whether CREB play a part role in the injury of pulmonarymicrocirculation barrier is unclear. The aim of this study is to investigate the role ofCREB in the injury of rat pulmonary microvascular endothelial cell (RPMVEC)induced by LPS.MethodsPrimary RPMVEC were successfully isolated and cultured in vitro, then treated withdifferent concentrations of LPS (0,0.1,1,10,100mg·L-1), or incubated for differenttimes (0,10,30,60,120min), or pre-incubated with cAMP-Dependent Protein KinasePeptide Inhibitor (PKI) for30min. Western-blot was used to assay the phosphorylationlevels of CREB, endothelial permeability was determined by measuring the influx ofEvans blue-labeled albumin (EBA) across endothelial monolayer.Results1) RPMVEC were isolated and cultured successfully in vitro, and were confirmed by morphology and fluorescein isothiocyanate-banderiraea simplicifolia I isolectin B4（FITC-BSI）integration experiment.2) In pace with increasing concentrations of LPS (0,0.1,1,10,100mg·L-1), thephosphorylation level of CREB at Ser-133was gradually increased in RPMVEC;LPS increased CREB phosphorylation at Ser133in RPMEC in a time-dependentmanner, peaked at30min, then gradually decreased, but still higher at120mincompared with control group.3) Western blot revealed that LPS also increased the expression of CREB in atime-dependent manner，raised at1h, peaked at3h, then gradually decreased, butstill higher at24h compared with control group.4) pre-incubated with cAMP-Dependent Protein Kinase Peptide Inhibitor (PKI) for30min., the CREB phosphorylation at Ser-133stimulated in the presence of LPSwere nearly suppressed in RPMVEC(P<0.05). Compared with control group,there is no significant decline in CREB phosphorylation at Ser-133stimulated withPKI (P＞0.05).5) Compared with control group, the monolayer permeability of RPMVECincubated with10mg·L-1LPS significantly increased (P<0.05); while pre-incubatedwith10μmol·L-1PKI for30min, the monolayer permeability of PMVECincreased significantly than10mg·L-1LPS stimulation group (P <0.05);compared with control group, PKI alone also increased the monolayer permeabilityof RPMVEC, the difference was statistically significant (P <0.05).Conclusions1) LPS rapidly induce the phosphorylation of CREB in RPMVEC in a dose-andtime-dependent manner, and PKA mediates the process.2) Phosphorylation of CREB may play a protective role in the process ofLPS-stimulated injury of RPMVEC.