摘要
目的探究黄芪甲苷对纤维状Aβ_(1-42)诱导的体外血脑屏障(BBB)模型损伤的影响及可能机制。方法制备Aβ_(1-42)诱导的小鼠脑微血管内皮细胞b End.3和原代大鼠星形胶质细胞As非接触式共培养BBB模型,分为对照组、模型组(Aβ_(1-42)30μmol/L)及黄芪甲苷低、高剂量(50、200μmol/L)组;利用噻唑蓝(MTT)法检测黄芪甲苷对Aβ_(1-42)诱导的b End.3细胞活力的影响;通过检测荧光素钠透过体外BBB模型的量鉴定BBB的通透性;Western blotting法检测细胞凋亡执行蛋白半胱天冬蛋白酶-3(Caspase-3)、活化的半胱天冬蛋白酶-3(cleaved Caspase-3)的表达水平以及BBB内皮细胞间相关连接蛋白细胞质透明带蛋白-1(ZO-1)、Claudin-5、Occludin的表达水平。结果 MTT结果显示,与模型组比较,黄芪甲苷低、高剂量组均能显著提高b End.3细胞活性(P<0.001),且保护作用与黄芪甲苷浓度呈正相关;荧光素钠通透性实验结果显示,与模型组相比较,黄芪甲苷预处理可显著降低BBB的通透性(P<0.001)。Western blotting结果显示,与模型组比较,黄芪甲苷预处理后,cleaved Caspase-3/Caspase-3显著降低,ZO-1、Claudin-5、Occludin蛋白的表达水平显著增加(P<0.001)。结论黄芪甲苷可能是通过抑制Aβ_(1-42)诱导的脑微血管内皮细胞b End.3的凋亡及增加其连接蛋白表达而发挥BBB保护作用。
Objective To explore the effect of astragaloside IV on the damaged blood-brain barrier（BBB） in vitro model induced by amyloid-beta protein（Aβ1-42） and the underlying mechanism. Methods Firstly, the non-contact co-culture blood-brain barrier（BBB） model was established by Aβ1-42-induced mouse brain microvascular endothelial cell（b End.3） and primary rat astrocyte（As）. Then the mice were divided into four groups： control, model（Aβ1-42 30 μmol/L）, astragaloside IV low and high dose groups（Aβ1-42 30 μmol/L with Astragaloside IV 50 and 200 μmol/L）. The effect of astragaloside IV on the vitality of b End.3 induced by Aβ1-42 was detected by methyl thiazolyl tetrazolium（MTT） assay. The permeability of BBB in vitro was determined by detecting the quantity of fluorescein sodium through BBB in various groups. In order to explore the mechanism of its protection, the apoptosis related proteins Caspase-3, cleaved Caspase-3 and tight junction proteins ZO-1, Claudin-5 and Occludin were detected by Western blotting. Results Compared with model group, the astragaloside IV groups improved the activity of b End.3 cells significantly（P〈0.001）. The protective effect was positively correlated with the concentration of astragaloside IV. Astragaloside IV with low and high dose decreased the permeability of BBB model in vitro（P〈0.001）. According to the results of Western blotting, the ratio of cleaved Caspase-3/Caspase-3 was significantly declined, and the expression levels of ZO-1, Claudin-5 and Occludin were significantly increased in astragaloside IV groups（P〈0.05）. Conclusion Astragaloside IV may play a BBB protective role by inhibiting the apoptosis of b End.3 cells induced by fibrous Aβ1-42 and increasing the expression of tight junction proteins.
作者
张乐裕
刘晨阳
于顾然
ZHANG Le-yu;LIU Chen-yang;YU Gu-ran(Jiangyin traditional Chinese medicine Hospital,Affiliated Hospital of Nanjing University of Traditional Chinese medicine,Jiangyin 214400,China)
出处
《中草药》
CAS
CSCD
北大核心
2018年第17期4099-4105,共7页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81573771)
江苏省自然科学基金项目(BK20151599)
