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目的:建立体外血脑屏障模型,研究补阳还五汤对氧糖剥夺再灌注损伤状态下血脑屏障功能及紧密连接相关蛋白表达的影响。方法:分离培养原代大鼠脑微血管内皮细胞,建立体外血脑屏障氧糖剥夺再灌注损伤模型,将细胞分为空白组、模型组、补阳还五汤提取液处理组,采用细胞计数试剂盒(CCK-8)法检测细胞活性;采用转移小室(Transwell)检测牛血清白蛋白(BSA)的透过量;蛋白质印迹(Western blot)实验检测紧密连接蛋白闭合蛋白(Occludin)和紧密连接相关蛋白-1(ZO-1)的表达;利用实时荧光定量聚合酶链式反应(q PCR)检测紧密连接蛋白Occludin,ZO-1 mRNA的表达。结果:与空白组比较,模型组细胞活力降低,BSA的透过率明显升高,Occludin,ZO-1蛋白及其mRNA的表达量明显降低(P<0.05);与模型组比较,补阳还五汤预处理后细胞存活性明显升高,BSA的透过率降低,Occludin,ZO-1蛋白及其mRNA的表达量明显升高(P<0.05)。结论:补阳还五汤能够降低氧糖剥夺再灌注处理对体外血脑屏障模型的损伤,上调紧密连接蛋白ZO-1,Occludin蛋白及其mRNA的表达量。
OBJECTIVE: To establish an in vitro blood-brain barrier model to study the effect of Buyang Huanwu Decoction on the function of the blood-brain barrier and the expression of tight junction-associated proteins under oxygen-glucose deprivation-reperfusion injury. Methods: Primary rat brain microvascular endothelial cells were isolated and cultured to establish an in vitro blood-brain barrier model of oxygen-glucose deprivation-reperfusion injury. The cells were divided into blank group, model group and BYHWD extract group. Cell counting reagents Cell viability was determined by CCK-8 assay. Bovine serum albumin (BSA) translocation was measured by Transwell assay. Occludin and tight junction-related proteins were detected by Western blot. 1 (ZO-1). The expression of tight junction protein Occludin and ZO-1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (qPCR). Results: Compared with the blank group, the viability of model group decreased, the transfection rate of BSA increased significantly, the expression of Occludin, ZO-1 protein and mRNA decreased significantly (P <0.05). Compared with model group, After pretreatment with Wu Tang, the cell viability was significantly increased and the transmissivity of BSA was decreased. The expressions of Occludin, ZO-1 protein and mRNA were significantly increased (P <0.05). Conclusion: Buyang Huanwu Decoction can reduce the damage of blood-brain barrier model induced by oxygen-glucose deprivation and reperfusion, and up-regulate the expressions of tight junction protein ZO-1 and Occludin.