对羟基苯甲醛对血脑屏障的保护作用及机制研究  

Protective effect and mechanism of p-hydroxybenzaldehyde on blood-brain barrier

作　　者：祝燕平[1,2] 李欣[1] 杜瑶[1] 张璐 冉利 周宁娜 ZHU Yan-ping1,2, LI Xin1 , DU Yao1 , ZHANG Lu1 , RAN Li1 , ZHOU Ning-na1.(1. Department of Pharmacology, Yunnan University of Traditional Chinese Medicine, Kunming 650500, 2. Department of Pharmacy, Anqing Medical College, Anqing 246052, Chin)

机构地区：[1]云南中医学院药理教研室,云南昆明650500 [2]安庆医药高等专科学校药学系,安徽安庆246052

出　　处：《中国中药杂志》2018年第5期1021-1027,共7页China Journal of Chinese Materia Medica

基　　金：云南省教育厅科学研究重大专项项目(ZD2015011);云南省应用基础研究计划重点项目(2016FA035);云南省中医药领军人才项目

摘　　要：脑缺血过程中由氧化应激导致血脑屏障（BBB）的破坏是引起继发性脑损伤的重要病理反应，该研究目的是探讨天麻酚性成分对羟基苯甲醛（p-hydroxybenzaldehyde，p-HBA）对BBB的保护作用及相关机制。BBB主要由血管内皮细胞和星形胶质细胞组成，故该实验以小鼠脑微细血管内皮细胞株（bEnd．3）和星形胶质细胞（astrocyte，Ast）作为BBB模型进行研究。用H2O2诱导bEnd．3和Ast氧化应激的方法复制BBB损伤模型。采用不同浓度H2O2（0．125，0．25，0．5，0．75mmol·L^-1）诱导bEnd．3和Ast损伤4h；再用0．5mmol·L^-1H202诱导bEnd．3及Ast损伤1，2，4，6h，检测乳酸脱氢酶（LDH）漏出率以筛选H2O2诱导细胞氧化损伤的最佳条件。不同浓度P-HBA（12．5，25，50mg·L^-1）干预后，检测bEnd．3和Ast细胞LDH漏出率；采用Westernblot法检测正常bEnd.3和Ast以及H2O2诱导损伤后Ast细胞中Nrf2，HO-1和NQ01蛋白表达量的变化；采用免疫荧光法和Westernblot法检测p-HBA对bEnd．3中紧密连接蛋白claudin-5和occludin表达的影响。结果显示，0．5rnmol·L^-1H202诱导bEnd．3和Ast损伤4h为最佳条件。不同浓度p-HBA均可降低H2O2诱导bEnd．3和Ast损伤后细胞LDH的漏出率；可增加bEnd．3中claudin-5，occludin，Nrt2，HO-1及NQ01蛋白的表达量；可增加Ast及H2O2诱导损伤后Ast中Nrf2，HO-1和NQ01蛋白表达量。结果提示，p-HBA可保护氧化应激诱导的BBB损伤，其机制与增加bEnd．3紧密连接蛋白claudin-5，OC-cludin的表达及激活bEnd-3和Ast细胞中抗氧化应激Nrt2／ARE通路、增强细胞内源性抗氧化能力有关。The disruption of blood-brain barrier （BBB） induced by oxidative stress is an important pathological reaction which re- suits in secondary brain injury during the cerebral ischemia-reperfusion. This study was designed to investigate the protective effect and mechanism of p-hydroxybenzaldehyde （p-HBA） from Gastrodia clara on BBB. The BBB is mainly consisted of vascular endothelial cells and astrocytes, so brain microvascular endothelial cell line （ bEnd. 3 ） and astrocytes （Ast） in mice were used in this study to establish BBB model. H202-induced oxidative stress was employed to induct the BBB damage. The bend. 3 cells or astroeytes were exposed to different concentrations of H202（0. 125, 0. 25, 0. 5,0. 75 mmol.L^-1 ） for4 h, then exposed to 0. 5 mmol.L-1 H202 for different du- ration （1,2, 4, 6 h） to detect the reasonable condition of oxidative injury. After intervention by different concentrations of p-HBA （ 12. 5, 25, and 50 mg·L^-1 ）, LDH leakage rate was detected for bend. 3 and Ast cells; the expression levels of tight junction protein claudin-5 and occludin in bend. 3 cells were determined by Western blot and immunofluorescenee. Nrf2, HO-1 and NQOI in normal bend. 3 cells and astrocytes as well as H202-induced damaged in astrocytes were detected by western blot after treatment with p-HBA. The results showed that the optimal condition of H202, induced damage in bend. 3 cells and astrocytes was set up as exposure the cells to 0. 5 mmol·L^-1 H202 for 4 h. Different concentrations ofp-HBA could decrease LDH leakage rate after bEnd. 3 and Ast injury was induced by H202 ; increase the protein expression levels of claudin-5, occludin, Nrf2, HO-1 and NQ01 ; and increase the expression levels of Nrf2, HO-1 and NQO1 in normal and H202-induced damaged astrocytes. These findings indicate that the p-HBA has protec- tive effect on the BBB, and the related mechanism seems to involve up-regulating tight junction protein of the bEnd. 3 cells and enhan- cing endogenous antioxidant capacity by ac

关 键 词：血脑屏障 氧化损伤 小鼠脑微细血管内皮细胞株 星形胶质细胞 对羟基苯甲醛 

分 类 号：R285.5[医药卫生—中药学]