基于斑马鱼模型研究木蝴蝶苷A的抗阿尔茨海默病活性和作用机制

Anti-Alzheimer′s disease activity of oroxin A and its mechanism of action based on zebrafish model

基于六水合氯化铝诱导的斑马鱼阿尔茨海默病模型,探究木蝴蝶苷A的抗阿尔茨海默病活性及作用机制。将受精后3 d的野生型AB品系斑马鱼随机分为阴性对照组,80μmol/L六水合氯化铝模型对照组,80μmol/L六水合氯化铝与6μmol/L多奈哌齐阳性对照组,80μmol/L六水合氯化铝与不同浓度(5、10、20μmol/L)木蝴蝶苷A受试物组。斑马鱼受精后6 d,利用明暗交替行为学实验观察不同处理组斑马鱼行为差异并分析其变化;通过硫黄素S染色测定各组斑马鱼头部Aβ斑块沉积数;采用酶活测定试剂盒检测各组斑马鱼乙酰胆碱酯酶活性;以实时荧光定量PCR检测自噬相关基因(beclin1、ulk1b、ulk2和atg7)的表达变化;借助分子对接技术验证木蝴蝶苷A与自噬相关蛋白(beclin1、ulk1b、ulk2和atg7)结合的可靠性。结果表明,木蝴蝶苷A缓解了六水合氯化铝造成的斑马鱼运动障碍,降低了Aβ斑块沉积数和乙酰胆碱酯酶活性水平,使自噬相关基因的异常表达趋于正常。该研究初步揭示了木蝴蝶苷A能够缓解六水合氯化铝诱导的斑马鱼运动障碍,其机制可能与激活细胞自噬有关,这为木蝴蝶苷A的临床应用及其治疗阿尔茨海默病的相关研究提供了理论依据。

To investigate the ameliorative effects of oroxin A on Alzheimer′s disease(AD)and the underlying mechanism of action,a zebrafish AD model induced by aluminum chloride hexahydrate(AlCl 3)was used.Wild-type zebrafish AB larvae at 3 dpf(days post fertilization)were divided into different groups,including negative control group,AlCl 3(80μmol/L)model control group,AlCl 3(80μmol/L)combined with donepezil(6μmol/L)positive control group,and AlCl 3(80μmol/L)combined with different concentrations(5,10,and 20μmol/L)of oroxin A test group.At 6 dpf,zebrafish behavior was monitored and analyzed using zebrafish light-dark locomotion test.Aβdeposition in zebrafish heads was assayed by thioflavin S staining.Acetylcholine assay kit tested acetylcholinesterase(AchE)activity.In addition,the expression of autophagy-related genes(beclin1、ulk1b、ulk2 and atg7)was tested by real-time quantitative polymerase chain reaction.Molecular docking was performed to validate the interaction between oroxin A and autophagy-related protein(beclin1、ulk1b、ulk2 and atg7).The results indicated that oroxin A significantly relieved the dyskinesia and inhibited Aβdeposition and AchE activity of zebrafish induced by AlCl 3.The expression of autophagy-related genes tended to be normal after oroxin A treatment.This study preliminarily revealed that oroxin A alleviated AlCl 3-induced AD symptoms in zebrafish,where the underlying mechanism of action is possibly associated with activated autophagy,providing a theoretical basis for the clinical application of oroxin A and its related research in treating AD.