文章摘要
顾晓群,余黎,武相,白宇,徐奚如,张静,周春梅,张彪.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[J].南京中医药大学学报,2020,36(1):56-62.
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
  
DOI:
中文关键词: 关键词:阿尔茨海默病  补肾益智方  斑马鱼  β淀粉样蛋白  氧化应激
英文关键词: Alzheimer's disease  Bushen Yizhi Formula  zebrafish  amyloid-beta  oxidative stress
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作者单位
顾晓群1,余黎2,武相1,白宇1,徐奚如1,张静1,周春梅1,张彪1 1.南京中医药大学附属医院/江苏省中医院江苏 南京 2100292.南京中医药大学药学院江苏 南京 210023 
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中文摘要:
      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。
英文摘要:
      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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