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bEnd.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 30 μmol/L）及黄芪甲苷低、高剂量（50、200 μmol/L）组；利用噻唑蓝（MTT）法检测黄芪甲苷对Aβ_1-42诱导的bEnd.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bEnd.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bEnd.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 (Aβ_1-42) and the underlying mechanism. Methods Firstly, the non-contact co-culture blood-brain barrier (BBB) model was established by Aβ_1-42-induced mouse brain microvascular endothelial cell (bEnd.3) and primary rat astrocyte (As). Then the mice were divided into four groups:control, model (Aβ_1-42 30 μmol/L), astragaloside IV low and high dose groups (Aβ_1-42 30 μmol/L with Astragaloside IV 50 and 200 μmol/L). The effect of astragaloside IV on the vitality of bEnd.3 induced by Aβ_1-42 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 bEnd.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 bEnd.3 cells induced by fibrous Aβ_1-42 and increasing the expression of tight junction proteins.

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）