p115RhoGEF/RhoA信号通路在高糖致脑微血管内皮细胞通透性异常中的作用

ROLE OF p115RhoGEF/RhoA SIGNALING PATHWAY IN PERMEABILITY OF BRAIN MICROVASCULAR ENDOTHELIAL CELLS EXPOSED TO HIGH GLUCOSE

目的探讨p115RhoGEF/RhoA信号通路在高浓度葡萄糖(Glucose)致小鼠脑微血管内皮细胞(bEnd.3通透性异常中的作用。方法体外培养小鼠脑微血管内皮细胞(bEnd.3),构建单层血脑屏障体外模型。通过ESR电阻仪测定跨内皮细胞间电阻(TEER),分光光度法检测碱性磷酸酶(ALP)、γ-谷胺酞胺转移酶(γ-GT)活性以评估其屏障功能;对照组(5.6mmol/L葡萄糖)、高糖诱导组(15.6、25.6、35.6、45.6mmol/L葡萄糖)处理细胞48h,MTT检测细胞相对存活率,分光光度法检测培养上清中乳酸脱氢酶(LDH)活性,免疫印迹法(Western blot)检测紧密连接蛋白ZO-1、Occludin表达水平,评估高糖对脑微血管内皮细胞通透性的影响;Western blot检测不同浓度葡萄糖处理细胞后p115RhoGEF蛋白表达水平,体外蛋白与蛋白结合试验(Pull-Down)检测RhoA活性。转染p115RhoGEF-siRNA建立p115RhoGEF基因沉默的bEnd.3细胞模型,检测不同处理组RhoA活性及p115RhoGEF、TJ蛋白表达水平。结果细胞TEER值随培养时间延长逐渐升高,ALP、γ-GT活力均随时间延长逐渐升高;与对照组比较,35.6、45.6mmo/L葡萄糖诱导组的细胞活力出现明显下降作用,且诱导时间越长,细胞存活率越低(P<0.05);35.6mmol/L葡萄糖诱导组的LDH水平明显升高(P<0.05);随着葡萄糖浓度升高,ZO-1、Occludin蛋白表达下降而p115RhoGEF蛋白表达升高,差异有统计学意义(P<0.05);Pull-down结果显示,25.6、35.6mmol/L葡萄糖组的RhoA活性较对照组明显升高(P<0.05)。细胞转染p115RhoGEF-siRNA后,p115RhoGEF蛋白表达较对照组明显下降;高糖诱导48h后,与NCsiRNA组比较,p115RhoGEF-siRNA组的GTP-RhoA活性下降、 ZO-1、 Occludin表达水平明显上调(P<0.05)。结论高糖通过诱导p115RhoGEF/RhoA信号通路活化,下调紧密连接蛋白表达,引起脑微血管内皮细胞通透性增加。

Objective To explore the role of p115 RhoGEF/RhoA signaling pathway in the permeability of brain microvascular endothelial cells(bEnd.3) exposed to high glucose. Methods To establish monolayer blood-brain barrier model(BBB) in vitro, mouse cerebral microvascular endothelial cell line(bEnd.3) were incubated from 1 to 10 days. Cell morphology alteration was observed under phase-contrast microscopy. The barrier integrity was measured by transendothelial electrical resistance(TEER) assay. γ-Glutamyl transpeptidase(γ-GT) and alkaline phosphatase(ALP) activity of the BBB were detected by spectrophotometry. bEnd.3 cells were treated with 5.6, 15.6, 25.6, 35.6,45.6 mmol/L glucose for 48 h. Cell viability was determined using an MTT assay. Lactate dehydrogenase(LDH) activity in the supernatant was determined by the spectrophotometry assay. The expressions of ZO-1, occludin and claudin-5 proteins were measured by Western blot analysis to evaluate the effect of glucose on tight junction proteins in bEnd.3 cells. Expression of p115 RhoGEF protein and RhoA activity were measured using Western blot analysis and pull-down assay, respectively. The p115 RhoGEF-siRNAbEnd.3 cell model was established by transfection of p115 RhoGEF-siRNA. Western blot analysis measured the expression of p115 RhoGEF and TJ protein, and pull-down assay measured the GTP-RhoA activity.Results TEER, activity of γ-GT and ALP were all increased with the culture time in bEnd.3 cell, indicating the in-vitro model had good barrier properties. MTT showed that cell viability decreased with increasing Glu concentrations(35.6, 45.6 mmol/L)(P<0.05). Especially, cell viability decreased after 35.6 mmol/L treatment in a time-dependent manner. The level of LDH significantly increased after the Glu treatment at 35.6 mmol/L. Exposure to Glu significantly increased the expression of occludin and ZO-1 protein,whereas no significant effect on the expression of claudin-5 was observed. The levels of p115 RhoGEF and RhoA were markedly increased after 48 h of Glu treatment(25.6, 35.6 mmol/L)(P<0.05).Transfection of p115 RhoGEF-siRNA effectively reduced the expression of p115 RhoGEF protein and RhoA activity in bEnd.3 cells exposed to high glucose(P<0.05). Loss of ZO-1 and occludin protein could be reversed by inhibition of p115 RhoGEF(P<0.05).Conclusion Exposure to high glucose down-regulates the expression of occludin and ZO-1 proteins and results in an increased permeability of bEnd.3 cells, which may be mediated by the p115 RhoGEF/RhoA signaling pathway. Transfection of p115 RhoGEF-siRNA plays a protective role in the permeability of brain microvascular endothelial cells by blocking the activation of p115 RhoGEF/RhoA signaling pathway.