脂多糖对大鼠脑微血管内皮细胞通透性的影响及机制研究

Influence of lipopolysaccharide on the permeability of rat brain microvascular endothelial cells and the molecular mechanism

摘要:

目的：了解脂多糖（LPS）对大鼠脑微血管内皮细胞(BMECs)通透性的影响及其可能的机制。方法：分离和培养原代大鼠BMECs，随机分为对照组和LPS干预组。利用跨内皮细胞电阻抗检测 BMECs 屏障功能，Pull-down法测定RhoA活性，Western blot检测p115RhoGEF和紧密连接蛋白ZO-1、occludin、claudin-5的蛋白表达量。结果：与对照组（159.0±8.6 Ω?cm2）比较，LPS作用3 h后，大鼠BMECs 跨内皮细胞电阻抗值明显下降(108.3±4.2 Ω?cm2)，作用12 h后达最低(85.4±2.5 Ω?cm2)。LPS作用5 min后RhoA明显活化，1 h后出现p115RhoGEF蛋白表达上调，3 h后紧密连接蛋白ZO-1、occludin和claudin-5的表达水平均不同程度下降，与对照组比较差异有统计学意义（P<0.05）。结论：LPS诱导大鼠BMECs中p115RhoGEF/RhoA信号通路活化，导致紧密连接蛋白表达水平降低，引起 BMECs 通透性增加。

Abstract:

OBJECTIVE: To study the influence of lipopolysaccharide (LPS) on the permeability of rat brain microvascular endothelial cells (BMECs) and possible molecular mechanism. METHODS: Monolayers of primary rat BMECs were separated and cultured, and then treated with (LPS group) or without LPS (control group). The barrier integrity was measured by transendothelial electrical resistance (TEER) assay. The degrees of RhoA activation were determined by Pull-down assay. The expression levels of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis. RESULTS: The average TEER values of rat BMECs in the LPS group were 108.3±4.2 Ω?cm2 and 85.4±2.5 Ω?cm2 respectively 3 and 12 hrs after LPS treatment, which were significantly lower than that in the control group (159.0±8.6 Ω?cm2). Compared with the control group, the activity of RhoA started to increase 5 minutes after LPS treatment, and the expression of p115RhoGEF protein started to increase 1 hr after LPS treatment and the cellular protein levels of ZO-1, occludin and claudin-5 decreased significantly 3 hrs after LPS treatment in the LPS group (P<0.05). CONCLUSIONS: LPS may activate the p115RhoGEF/RhoA pathway and decrease protein expression of ZO-1, occludin and claudin-5, resulting in an increased permeability of rat BMECs.