补肾益智方治疗阿尔茨海默病斑马鱼模型的作用及机制研究

Research on the Effect and Mechanism of Bushen Yizhi Formula Treating Alzheimer's Zebrafish Model

    摘要
  • 摘要: 目的 通过阿尔茨海默病斑马鱼模型，探讨补肾益智方的作用机制。方法 选取6月龄斑马鱼，随机分成5组，分为空白对照组（n=12）、模型组（n=10）、阳性药组（n=10）、补肾益智方高剂量组（n=11）和低剂量组（n=12）。除空白对照组，其余各组均用AlCl3·6H2O配制成浓度为100 μg/L的溶液持续浸泡斑马鱼30 d，每天更换一半的染毒培养液。染毒结束后利用T迷宫实验剔除染毒失败的斑马鱼。造模结束后，分别给予阳性药组多奈哌齐20 μg/mL，高、低剂量组补肾益智方30、10 μg/mL浸泡14 d。给药结束后，通过T迷宫实验检测斑马鱼学习记忆能力；免疫组化染色观察神经元变化；qPCR检测相关基因的表达；Western blot检测相关蛋白的表达。结果 ①在T迷宫测试第4天，模型组潜伏时间为（198.00±45.78）s，补肾益智方低剂量组为（12.75±2.29）s，补肾益智方高剂量组为（7.27±0.90）s，补肾益智方治疗组斑马鱼的潜伏时间明显低于模型组，差异有统计学意义（P<0.05）；②补肾益智方治疗组斑马鱼的端脑神经元数量较模型组明显增多、排列变整齐，星型胶质细胞增多；③补肾益智方组斑马鱼脑ACHE活力显著降低（P<0.01）、ChAT活力显著增加（P<0.05）；④补肾益智方治疗组斑马鱼脑组织中appb、bace1表达显著降低（P<0.01），sod、cat、nrf2基因表达显著增加（P<0.01），keap1基因表达显著降低（P<0.01）；⑤补肾益智方治疗组斑马鱼脑组织蛋白表达情况与qPCR结果一致，APP、BACE1表达显著降低（P<0.01），Nrf2表达显著增加（P<0.01），Keap1表达显著降低（P<0.01）。结论 补肾益智方能有效改善AD斑马鱼的学习记忆能力，减少Aβ生成，减轻氧化应激。

     

    Abstract: OBJECTIVE To explore the changes in learning-memorial ability and a series of targets by Bushen Yizhi Formula in zebrafish. METHODS Six months old zebrafish were randomly divided into 5 groups: control group(n=12)，model group(n=10)，positive drug group(n=10)，Bushen Yizhi Formula high dose group(n=11)，Bushen Yizhi Formula low dose group(n=12). Each group， except the control group， was placed in AlCl3·6H2O with a concentration of 100 μg/L for 30 days and replace half of the culture solution every day. After contamination， the positive drug group was treated with Donepezil in 20 μg/mL， the Bushen Yizhi Formula high dose group was treated with herbs in 30 μg/mL， the Bushen Yizhi Formula low dose group was treated in 10 μg/mL for 14 days. After the drug intervention， learning and memory ability were detected by T-maze behavior examination; The changes of nerve cells were observed by immunohistochemistry; The expression of gene by qPCR; The expression of protein by Western blot. RESULTS ①In the fourth day of T-maze behavior training， the incubation period of model group， Bushen Yizhi Formula low dose group and Bushen Yizhi Formula high dose group were (198.00±45.78)s， (12.75±2.29)s， (7.27±0.90)s， respectively. The incubation period of Bushen Yizhi Formula group was significantly lower than that of the model group (P<0.05). ②The number of nerve cells and astrocyte cells in telencephalon of zebrafish were significantly greater in the treatment group than that in the model group. ③The activity of ACHE was lower and the activity of ChAT was higher in the treatment group than that in the model group. ④Comparing with the model group， the expression of appb， bace1， keap1 (P<0.01) in the brain of zebrafish which were treated by Bushen Yizhi Formula significantly reduced， and the expression of sod， cat， nrf2(P<0.01) increased. ⑤The results of Western blot were consistent with the results of qPCR. The expressions of APP and BACE1 significantly reduced (P<0.05)， Nrf2 significantly increased (P<0.01)， and Keap1 significantly reduced (P<0.01). CONCLUSION Bushen Yizhi Formula can improve the learning-memorial ability， reduce the production of Aβ and oxidative stress in zebrafish.

     

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