异丙酚在血脑屏障氧糖剥夺体外模型中对紧密连接蛋白ZO-1表达的影响

Effect of Propofol on tight junction protein ZO-1 in glucose of in vitro oxygen deprivation brain-blood barrier model

目的探讨异丙酚在血脑屏障氧糖剥夺(OGD)体外模型中对紧密连接蛋白ZO-1表达的影响及其机制。方法分离纯化Bal b/c小鼠的脑微血管内皮细胞进行体外培养,构建血脑屏障体外模型,并通过OGD处理模拟体内缺氧、缺血状态将细胞分为对照组、OGD损伤组、不同浓度(5、10、20、40μmol/L)异丙酚处理组及蛋白激酶C(PKC)激活剂佛波酯(TPA)处理组。四甲基偶氮唑盐(MTT)法检测内皮细胞存活力,电阻仪检测内皮细胞阻抗(TEER)值,聚合酶链反应(PCR)和Western blot分别检测异丙酚对ZO-1 mRNA和蛋白表达的影响,PKC活性检测试剂盒测定PKC活性;通过加入PKC激活剂检测PKC在异丙酚调节ZO-1表达过程中的作用。结果 10、20、40μmol/L异丙酚可显著抑制OGD诱导的内皮细胞活力,TEER值降低和ZO-1表达水平下降(P<0.05);此外,10、20、40μmol/L异丙酚还可抑制OGD诱导的PKC激活(0.856±0.124、1.017±0.104、0.930±0.149 vs 0.595±0.112)。在PKC激活剂的作用下,异丙酚对ZO-1 mRNA和蛋白表达的上调作用均无显著影响(0.361±0.441 vs 0.344±0.023,P=0.403;0.169±0.011 vs 0.161±0.019,P=0.489)。结论异丙酚可通过抑制PKC信号通路上调血脑屏障OGD体外模型紧密连接蛋白ZO-1的表达。

Objective To discuss the mechanism of Propofol on tight junction protein ZO-1 of the brain-blood barrier（BBB） under glucose and oxygen deprivation（OGD） conditions in vitro. Methods Mouse brain microvascular endothelial cells（MBVEC） were obtained from Bal b/c mouse and in vitro BBB was established. In OGD mimic state of hypoxia ischemia in vivo, cells were divided into control, OGD injured, different concentration（5, 10, 20, 40 μmol/L） of Propofol treated and protein kinase C（PKC） inhibitor 12- O-tetradecanoylphorbol 13-acetate （TPA） treated groups. MTT assay was used to detect MBVEC viability; and transendothelial electric resistance （TEER） was measured by resistance meter. Effects of Propofol on ZO-1 mRNA and protein expression were detected by PCR and Western blot, respectively, and the role of PKC in process of Propofol affecting ZO-1 was tested by PKC activator. Results The 10, 20, 40 μmol/L Propofol could significantly inhibited endothelial cells viability, TEER and expression of ZO-1 decreased（P〈 0.05）. The activity of PKC was significantly enhanced by OGD（0.856 ± 0.124, 1.017 ±0.104, 0.930 ± 0.149 vs 0.595 ± 0.112）. After the treatment of PKC activator, the up-regulate effect of Propofol on ZO-1 was inhibited in BBB of OGD in vivo （0.361 ± 0.441 vs 0.344± 0.023, P = 0.403; 0.169 ± 0.011 vs 0.161 ± 0.019, P = 0.489）. Conclusion It is demonstrated that PKC can up-regulate expression of tight junction protein ZO-1 by PKC signal pathway in OGD of BBB model in vivo.