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100μg/L的溶液持续浸泡斑马鱼30d,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20μg/mL,高、低剂量组补肾益智方30、10μg/mL浸泡14d。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。
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 100μg/L for 30 days and replace half of the culture solution every day.After contamination,the positive drug group was treated with Donepezil in20μg/mL,the Bushen Yizhi Formula high dose group was treated with herbs in 30μg/mL,the Bushen Yizhi Formula low dose group was treated in 10μg/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 Aβand oxidative stress in zebrafish.
作者
顾晓群
余黎
武相
白宇
徐奚如
张静
周春梅
张彪
GU Xiao-qun;YU Li;WU Xiang;BAI Yu;XU Xi-ru;ZHANG Jing;ZHOU Chun-mei;ZHANG Biao(Jiangsu ProvincE-Hospital of ChinesE-Medicine,Affiliated to Nanjing University of ChinesE-Medicine,Nanjing,210029,China;School of Pharmacy,Nanjing University of ChinesE-Medicine,Nanjing,210023,China)
出处
《南京中医药大学学报》
CAS
CSCD
北大核心
2020年第1期56-62,共7页
Journal of Nanjing University of Traditional Chinese Medicine
基金
江苏省干部保健课题(BJ16018)
江苏省方剂高技术研究重点实验室/江苏省中药资源产业化过程协同创新中心资助项目(FJGJS201507)
江苏省中医院创新发展基金专项课题(Y2019CX42)
