麝香黄芪复方滴丸对体外缺血缺氧血脑屏障通透性及相关调节蛋白的影响

Effect of Compound Shexiang Huangqi Dropping Pills(麝香黄芪复方滴丸)on Ischemia and Hypoxia Injury in Vitro Blood-Brain Barrier and Related Regulatory Proteins

目的探究麝香黄芪复方滴丸调控体外缺血缺氧性血脑屏障(blood-brain barrier,BBB)模型的作用机制。方法利用大鼠脑微血管内皮细胞(brain microvessel endothelial cells,BMEC)与星形胶质细胞(astrocytes,AS)共培养建立体外BBB模型及缺血缺氧BBB模型。随机分为正常对照组、模型组、麝香黄芪复方滴丸高剂量组(500μg/mL)、中剂量组(100μg/mL)、低剂量组(10μg/mL)及依达拉奉对照组。通过跨内膜电阻(TEER)、辣根过氧化物酶法(HRP)检测BBB通透性变化情况。Western Blot法检测紧密连接(tight junction,TJ)相关蛋白闭合蛋白-5(Claudin-5)、连接黏附分子-A(junctional adhesion molecule,JAM-A)、跨膜蛋白的咬合蛋白(Occludin)和闭锁小带蛋白-1(zonula Occludens,ZO-1)表达水平。结果TEER及HRP实验显示,与正常对照组相比,模型组血脑屏障通透性增加(P<0.05);麝香黄芪复方滴丸高、中、低剂量组和依达拉奉组1 d、2 d、3 d各组通透性明显降低(P<0.05);WB结果显示,模型组Claudin-5、JAM-A、Occludin、ZO-1蛋白表达较正常组显著降低(P<0.05);麝香黄芪复方滴丸高、中剂量组较模型组Claudin-5、JAM-A、Occludin、ZO-1蛋白表达升高(P<0.05)。结论麝香黄芪复方滴丸能降低缺血缺氧性BBB模型的通透性,其作用机制与调控紧密连接蛋白而达到修复BBB的作用有关。

Objective To explore the mechanism of Compound Shexiang Huangqi Dropping Pills(麝香黄芪复方滴丸,SXHQP)regulating I/R injury BBB model in vitro.Methods Rat brain microvascular endothelial cells(BMEC)and astrocytes(AS)were co-cultured to establish BBB model in vitro.They were randomly divided into normal group,model group,SXHQP high dose group(500μg/mL),SXHQP medium dose group(100μg/mL),SXHQP low dose group(10μg/mL)and Edaravone group.The transmembrane resistance(TEER)and horseradish peroxidase(HRP)were used to detect BBB permeability.The expressions of claudin-5,junctional adhesion molecule-A(JAM-A),Occludin and Zonula Occludens(ZO-1)were detected by Western blot.Results TEER and HRP experiments showed that the blood-brain barrier permeability in the model group was increased compared with that of the normal group(P<0.05).The permeability of the SXHQP high,medium and low dose groups and the Edaravone group on 1 d,2 d and 3 d was significantly decreased(P<0.05).The expressions of Claudin-5,Jam-A,Occludin,and ZO-1 in the model group were significantly decreased compared with those of the normal group(P<0.05).The expressions of Claudin-5,Jam-A,Occludin and ZO-1 in the high and medium dose groups were higher than those in the model group(P<0.05).Conclusion SXHQP can reduce the permeability of I/R injury BBB model,and the mechanism may be related to the regulation of tight junction proteins.