摘要
目的:本研究旨通过生物信息学和细胞实验研究,揭示活血荣络方治疗阿尔兹海默病的分子机制。方法:通过GeneCards、String、DAVID数据库预测AD疾病潜在靶点,并对潜在靶点进行"蛋白-蛋白"互作网络(PPI)、GO分析,绘制"靶点-GO"热图。同时采用体外Aβ25-35损伤星形胶质细胞诱导AD细胞模型,并随机分为6组,分别为空白组、多奈哌齐组、sFRP、0.5倍活血荣络方组、1倍活血荣络方组、2倍活血荣络方组组,加相应刺激分别处理12 h、24 h、48 h、72 h。采用MTT法检测细胞活性、免疫组化法检测APP、Aβ蛋白定位、定量表达,以初步验证生物信息学预测结果。结果:收集筛选出AD共35个靶点,其中APP、APOE、PSEN1、PSEN2、SNCA相互作用明显,且APP、APOE在GO中显著富集。MTT结果显示各组OD值随着时间的延长而增高,各药物组细胞活性均高于空白组,其中2倍组细胞活性最高;sFRP组低于空白组。免疫组化结果显示药物组Aβ及APP的表达均低于空白组,2倍活血荣络方组均低于其它给药组,并且各组Aβ及APP表达随时间延长有逐渐减少趋势,但2倍活血荣络方组减少最明显。结论:基于"阴虚血瘀-荣气虚滞"理论立方的活血荣络方可能通过多靶点、多成分、多通路发挥治疗AD疾病的作用,并进一步实验验证其治疗作用可能与抑制APP、Aβ蛋白的表达,降低AChE活性,从而改善神经突触炎症毒性有关。
Objective: To reveal the molecular mechanism of Huoxue Rongluo Formula in the treatment of Alzheimer’s disease(AD) through bioinformatics and cellular experimental studies. Methods: The potential targets of AD were predicted by GeneCards, String and DAVID databases, and the potential targets were analyzed by "protein-protein" interaction network(PPI) and GO, and the "target-GO" heat map was drawn up. At the same time, the AD cell model was induced by Aβ25-35 murine injured astrocytes in vitro, and were randomly divided into the blank group, Donepezil group, sFRP group, 0.5 x Huoxue Rongluo Formula group, 1 x Huoxue Rongluo Formula group and 2 x Huoxue Rongluo Formula group. The rats were treated with corresponding stimulation for 12 h, 24 h, 48 h and 72 h respectively. The cell viability was detected by MTT method, and the localization and quantitative expressions of APP and Aβ protein were detected by immunohistochemical method, in order to verify the prediction results of bioinformatics. Results: A total of 35 AD targets were collected and screened, among which APP, APOE, PSEN1, PSEN2 and SNCA had obvious interaction, and APP and APOE were significantly enriched in GO. The results of MTT showed that the OD value of each group increased with time. The cell activity of each drug group was higher than that of the blank group, and the cell activity of the 2 x Huoxue Rongluo Formula group was the highest, whereas that of the sFRP group was lower than that of the blank group. The results of immunohistochemistry showed that the expressions of Aβ and APP in the drug groups were lower than those in the blank group, and that in the 2 x Huoxue Rongluo Formula group was lower than that in other groups;the expressions of Aβ and APP in each group gradually decreased with time, but the decrease in the 2 x Huoxue Rongluo Formula group was the most obvious. Conclusion: Based on the theory of "yin deficiency and blood stasis-stagnation of deficient nutritive qi", Huoxue Rongluo Formula may play a role in the treatment of AD through multi-target, multi-component and multi-pathway. Further experiments show that its therapeutic effect may be related to the inhibition of protein expressions of APP and Aβ and reducing the activity of AChE, thus to improve synaptic inflammatory toxicity.
作者
陈瑶
宋洋
杨仁义
杨小钰
胡华
刘利娟
周德生
CHEN Yao;SONG Yang;YANG Renyi;YANG Xiaoyu;HU Hua;LIU Lijuan;ZHOU Desheng(Department of Neurology,The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Hunan University of Chinese Medicine^Changsha 410208,China)
出处
《中医药学报》
CAS
2020年第9期21-26,共6页
Acta Chinese Medicine and Pharmacology
基金
国家自然科学基金项目(81874463)
湖南省科技厅科技创新平台与人才计划(2017SK4005)
湖南省中医药管理局资助项目(201824)
湖南省教育厅一般项目(18C0404)
湖南中医药大学中西医结合一流学科开放基金项目(2018ZXYJH13)。
