环孢素A通过下调NADPH氧化酶4改善慢性脑低灌注大鼠空间记忆能力

Cyclosporine A improves spatial memory following chronic cerebral hypoperfusion in rats by down-regulation of NADPH oxidase 4

目的观察环孢素A对慢性脑低灌注大鼠空间记忆的保护作用及其可能机制。 方法将60只SD大鼠随机分为假手术组、赋形剂组以及环孢素A干预小剂量组、中剂量组和大剂量组。采用双侧颈总动脉永久结扎法制备慢性脑低灌注模型。模型制作后第46天起，假手术组和赋形剂组给予橄榄油1 ml/d灌胃，小剂量组、中剂量组和大剂量环孢素组分别给予环孢素A 3 mg/kg、6 mg/kg和12 mg/kg灌胃，1次/d，连用14 d。采用Morris水迷宫实验检测空间记忆能力，采用逆转录聚合酶链反应（reverse transcriptase polymerase chain reaction, RT-PCR）检测大脑皮质NADPH氧化酶4（NADPH oxidase 4, NOX4）mRNA表达，免疫组化染色和蛋白质印迹法检测大脑皮质NOX4蛋白表达。 结果Morris水迷宫实验显示，各环孢素组逃避潜伏期均显著短于赋形剂组（P均〈0.05）。免疫组化染色显示，假手术组、赋形剂组、小剂量、中剂量和大剂量环孢素A组NOX4阳性细胞百分比分别为（4.43±0.37）%、（37.44±4.76）%、（18.05±2.91）%、（12.51±3.4）%和（11.06±1.74）%（F＝262.021，P〈0.001），其中赋形剂组显著高于假手术组（P〈0.01），各环孢素A组均显著少于赋形剂组（P均〈0.01）。RT-PCR显示，假手术组、赋形剂组、小剂量、中剂量和大剂量环孢素A组大脑皮质NOX4 mRNA相对表达水平分别为0.36±0.03、1.04±0.04、0.58±0.02、0.49±0.01和0.40±0.02（F＝1 324.941，P〈0.001），各环孢素A组均显著低于赋形剂组（P均〈0.01）。蛋白质印迹分析显示，假手术组、赋形剂组、小剂量、中剂量和大剂量环孢素A组大脑皮质NOX4蛋白表达水平分别为0.02±0.01、0.27±0.04、0.09±0.02、0.06±0.02和0.06±0.01（F＝222.692，P〈0.001），各环孢素A组均显著低于赋形剂组（P均〈0.01）。 结论环孢素A可能通过下调NOX4改善慢性脑低灌注大鼠空间记忆能力。

ObjectiveTo investigate the protection effect of cyclosporine A on spatial memory following chronic cerebral hypoperfusion in rats and its possible mechanism. MethodsSixty SD rats were randomly divided into sham operation, vehicle, low-dose cyclosporine A, medium-dose cyclosporine A, and high-dose cyclosporine A groups. A chronic cerebral hypoperfusion model was prepared by permanent bilateral ligation of bilateral common carotid arteries. From 46 days after modeling, olive oil 1 ml/d was used for intragastric administration in the sham-operation group and the vehicle group. Cyclosporine A 3 mg/kg, 6 mg/kg, and 12 mg/kg were administrated intragastrically in the low-dose, medium-dose and high-dose cyclosporine A groups, respectively, once a day for 14 days. The spatial memory was assessed using Morris water maze test. Reverse transcriptase polymerase chain reaction （RT-PCR） was used to detect the expression of NADPH oxidase 4 （NOX4） mRNA in the cerebral cortex. Immunohistochemical staining and Western blot were used to detect the expression of NOX4 protein in the cerebral cortex. ResultsThe Morris water maze test showed that the escape latencies in all cyclosporine A groups were significantly shorter than the vehicle group （all P〈0.05）. Immunohistochemical staining showed that the percentages of the NOX4-positive cells in the sham-operation, vehicle, low-dose, medium-dose, and high-dose cyclosporine A groups were 4.43%±0.37%, 37.44%±4.76%, 18.05%±2.91%, 12.51%±3.4%, and 11.06%±1.74%, respectively （F=262.021, P〈0.001）, the vehicle group was significantly higher than the sham-operation group （P〈0.01）, and all cyclosporine A groups were significantly less than the vehicle group （all P〈0.01）. RT-PCR showed that the expression levels of NOX4 mRNA in cerebral cortex in the sham-operation, vehicle, low-dose, medium-dose, and high-dose cyclosporine A groups were 0.36±0.03, 1.04±0.04, 0.58±0.02, 0.49±0.01, and 0.40±0.02, respectively （F=1 324.941, P〈0.001）, all cyclosporine A groups were significantly lower than the vehicle group （all P〈0.01）. Western blot showed that the expression levels of NOX4 protein in cerebral cortex in the sham-operation, vehicle, low-dose, medium-dose, and high-dose cyclosporine A groups were 0.02±0.01, 0.27±0.04, 0.09±0.02, 0.06±0.02, and 0.06±0.01, respectively （F=222.692, P〈0.001）, all cyclosporine A groups was significantly lower than the vehicle group （all P〈0.01）. ConclusionCyclosporine A may improve spatial memory following chronic cerebral hypoperfusion in rats by down-regulation of NOX4.