中文摘要：

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3h后,bEnd.3细胞TEER值明显下降[（53.67±2.01）Ω·cm2],作用12h后达最低[（37.67±3.05）Ω·cm2],3,6,12h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3,12h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0.5h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3h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。

英文摘要：

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 Rhodaminephalloidin staining. Results Incubation of monolayers with LPS caused substantial barrier hyperpermeability. Under the normal condition,the average TEER of bEnd. 3 was （ 82. 33 ±3. 11） Ω·cm2, while it decreased apparently to （ 53. 67 ±2. 01） Ω·cm2 and （ 37. 67 ±3. 05） Ω·cm2 when the cells 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.