CAJ | 학술논문

目的:探讨脂多糖(lipopolysaccharide,LPS)对小鼠脑微血管内皮细胞通透性的影响及其可能的分子机制.方法:以培养至融合的小鼠脑微血管内皮细胞株bEnd.3为实验对象,利用跨内皮细胞电阻抗(transendothelial electrical resistance,TEER)检测LPS对bEnd.3细胞屏障功能的改变及C3转移酶预处理后对其的影响.Pull down法和报告基因法分别测定RhoA和NF-κB活性.直接荧光染色法观察bEnd.3细胞F-actin的分布变化.结果:LPS作用3 h后,bEnd.3细胞TEER值明显下降[(53.67±2.01) Ω·cm2],作用12 h后达最低[(37.67±3.05) Ω·cm2],3,6,12 h组与正常对照组比较差异均有统计学意义(P＜0.05).C3转移酶预处理后,LPS作用3,12 h时细胞TEER值分别为(60.67±4.05) Ω·cm2和(40.60±2.33) Ω·cm2,均比相应时间点未预处理细胞的TEER值高,差异有统计学意义(P＜0.05).LPS作用bEnd.3细胞0.5 h后RhoA明显活化,NF-κB活性增加,C3转移酶预处理后其NF-κB活性明显下降,差异有统计学意义(P＜0.05).直接荧光染色发现在LPS作用3 h后细胞出现明显的F-actin重构,其程度随LPS作用时间的延长而加重,而C3转移酶预处理细胞F-actin重构发生较晚且程度较轻.结论:LPS通过使F-actin重组增加脑微血管内皮细胞的通透性,RhoA和NF-κB均参与此过程,且NF-κB信号分子受RhoA调控.
목적:탐토지다당(lipopolysaccharide,LPS)대소서뇌미혈관내피세포통투성적영향급기가능적분자궤제.방법:이배양지융합적소서뇌미혈관내피세포주bEnd.3위실험대상,이용과내피세포전조항(transendothelial electrical resistance,TEER)검측LPS대bEnd.3세포병장공능적개변급C3전이매예처리후대기적영향.Pull down법화보고기인법분별측정RhoA화NF-κB활성.직접형광염색법관찰bEnd.3세포F-actin적분포변화.결과:LPS작용3 h후,bEnd.3세포TEER치명현하강[(53.67±2.01) Ω·cm2],작용12 h후체최저[(37.67±3.05) Ω·cm2],3,6,12 h조여정상대조조비교차이균유통계학의의(P＜0.05).C3전이매예처리후,LPS작용3,12 h시세포TEER치분별위(60.67±4.05) Ω·cm2화(40.60±2.33) Ω·cm2,균비상응시간점미예처리세포적TEER치고,차이유통계학의의(P＜0.05).LPS작용bEnd.3세포0.5 h후RhoA명현활화,NF-κB활성증가,C3전이매예처리후기NF-κB활성명현하강,차이유통계학의의(P＜0.05).직접형광염색발현재LPS작용3 h후세포출현명현적F-actin중구,기정도수LPS작용시간적연장이가중,이C3전이매예처리세포F-actin중구발생교만차정도교경.결론:LPS통과사F-actin중조증가뇌미혈관내피세포적통투성,RhoA화NF-κB균삼여차과정,차NF-κB신호분자수RhoA조공.
Objective To investigate the molecular mechanism for change in permeability in brain microvascular endothelial cells (bEnd.3) induced by lipopolysaccharide (LPS). Methods Monolayers of bEnd.3 were exposed to LPS,in the presence or absence of exoenzyme C3 transferase. We monitored the monolayer barrier integrity by transendothelial electrical resistance assay (TEER),activity of RhoA by pull down assay,NF-κB by luciferase reporter assay,and F-actin dynamic structure by Rhodamine-phalloidin staining. Results Incubation of monolayers with LPS caused substantial barrier hyperpermeability. Under the had been treated for 3 and 12 h with LPS (P＜0.05). Such effects could be inhibited partly by pretreatment of RhoA inhibitor exoenzyme C3 transferase. LPS activated RhoA and NF-κB at 0.5 h. The C3 transferase could significantly reverse the NF-κB activation (P＜0.05). The F-actin rearrangments displayed in a time-dependent manner and occurred originally after the stimulation of LPS for 3 h,which could be diluted by the pretreatment of C3 transferase as well. Conclusion LPS induces the disruption of F-actin cytoskeleton and brain microvascular endothelial barrier integrity,in part,through RhoA and NF-κB activation. The mechanism underlying this pathophysiological effect of RhoA is to influence the disruption of the F-actin cytoskeleton by regulating NF-κB activites.