GW0742对凝血酶诱导的体外血脑屏障破坏的保护作用  

Protective effect of GW0742 on thrombine-induced destruction of the blood-brain barrier in vitro

作　　者：汪朝云 陈琴 王筱雅 熊英 张金娟 张晴 冯占辉 叶兰 Wang Zhao-yun;Chen Qin;Wang Xiao-ya;Xiong Ying;Zhang Jin-juan;Zhang Qing;Feng Zhan-hui;Ye Lan(Department of Pharmacology,School of Basic Medicine,Guizhou Medical University,Guiyang,Guizhou 550025,China;Laboratory of Multimedia Functionality,School of Basic Medicine,Guizhou Medical University,Guiyang,Guizhou 550025,China;Department of Neurology,Affiliated Hospital of Guizhou Medical University,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州医科大学基础医学院药理学教研室,贵州贵阳550025 [2]贵州医科大学基础医学院多媒体机能学实验室,贵州贵阳550025 [3]贵州医科大学附属医院神经内科,贵州贵阳550025

出　　处：《中国现代医学杂志》2024年第3期34-41,共8页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:81960224)。

摘　　要：目的探讨GW0742对凝血酶诱导的体外血脑屏障破坏的保护作用。方法选取新生3~7日龄的SD大鼠若干,提取原代脑微血管内皮细胞(BMEC)及星形胶质细胞(AC)。于体外构建BMEC单层培养模型和BMEC+AC共培养模型,分为BMEC单层培养模型组、BMEC+AC共培养组及Blank组(只加培养基);取体外血脑屏障(BBB)共培养模型,分为Control组和脑出血组(ICH组),Control组加入完全培养基处理,ICH组加入40 u/mL凝血酶处理,分别处理12 h模拟体外ICH模型,并通过4 h渗漏试验、荧光素钠通透性试验评价BBB及ICH模型的通透性。另取BBB共培养模型大鼠分成5个组:Control组、ICH组,以及GW0742低、中、高剂量组(1.25、2.50、5.00μmol/L),给药24 h后采用Western blotting检测MMP-9、ZO-1、Occludin蛋白表达。结果4 h渗漏试验结果显示:与Blank组比较,BMEC单层培养组能维持一定的液面差;与BMEC单层培养组比较,BMEC+AC共培养组能维持较好的液面差。荧光素钠通透性试验结果显示各组通透性为:Blank组>BMEC单层培养组>BMEC+AC共培养组。荧光素钠通透性试验结果显示:与Blank组比较,BMEC单层培养组在0.5、1.0、2.0 h时通透性降低(P<0.05);与BMEC单层培养组比较,BMEC+AC共培养组在0.5、1.0、2.0 h时通透性降低(P<0.05)。4 h渗漏试验结果显示:与Control组相比,ICH组液面差降低,通透性增加。荧光素钠通透性试验结果显示:与Control组比较,ICH组在0.5、1.0、2.0 h时通透性增加(P<0.05)。细胞形态学观察结果显示:与Control组比较,ICH组内皮细胞形态明显皱缩、拉长;与ICH组比较,给药组内皮细胞形态未见明显皱缩、拉长,呈剂量依赖。与Control组比较,ICH组MMP-9蛋白相对表达量升高(P<0.05),ZO-1、Occludin蛋白相对表达量降低(P<0.05);与ICH组比较,GW0742高剂量组MMP-9蛋白相对表达量降低(P<0.05),ZO-1、Occludin蛋白相对表达量升高(P<0.05)。结论GW0742对凝血酶诱导的血脑屏障破坏具有保护作用,�Objective To investigate the protective effects of GW0742 against thrombin-induced in vitro blood-brain barrier(BBB)disruption.Methods Neonatal SD rats aged 3 to 7 days were utilized to extract primary brain microvascular endothelial cells(BMEC)and astrocytes(AC).In vitro,BMEC monolayer culture and BMEC+AC co-culture models were established,including the BMEC monolayer culture model group,BMEC+AC co-culture group,and Blank group(culture medium only).For the in vitro BBB co-culture model,rats were assigned to the Control group and intracerebral hemorrhage(ICH)group.The Control group received complete culture medium,while the ICH group was treated with 40 u/mL thrombin for 12 hours to simulate the in vitro ICH model.BBB and ICH model permeability were evaluated through a 4-hour leakage test and sodium fluorescein permeability test.Rats in the BBB co-culture model were divided into five groups:Control group,ICH group,and GW0742 low,medium,and high dose groups(1.25,2.50 and 5.00μmol/L).After 24 hours of administration,Western blotting was used to detect MMP-9,ZO-1,and Occludin protein expressions.Results The 4-hour leakage test results showed that compared to the Blank group,the BMEC monolayer culture group maintained a certain fluid level difference,and the BMEC+AC co-culture group maintained a better fluid level difference than the BMEC monolayer culture group.The sodium fluorescein permeability test results demonstrated the following permeability order:Blank group>BMEC monolayer culture group>BMEC+AC co-culture group.In the sodium fluorescein permeability test,the BMEC monolayer culture group showed reduced permeability at 0.5,1.0,and 2.0 hours compared to the Blank group(P<0.05),and the BMEC+AC co-culture group exhibited decreased permeability at 0.5,1.0,and 2.0 hours compared to the BMEC monolayer culture group(P<0.05).The 4-hour leakage test results revealed that the ICH group had a decreased fluid level difference and increased permeability compared to the Control group.In the sodium fluorescein permeability

关 键 词：脑出血 血脑屏障 GW0742 MMP-9 ZO-1 OCCLUDIN 

分 类 号：R743.34[医药卫生—神经病学与精神病学]