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

doi:10.3976/j.issn.1002-4026.2023.06.005

山东科学 ›› 2023, Vol. 36 ›› Issue (6): 28-37.doi: 10.3976/j.issn.1002-4026.2023.06.005

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

时瑞碟¹^,²(), 高鑫², 王宝堃², 高代丽², 靳梦²^,^*(), 张秀军¹^,^*()

1.华北理工大学心理与精神卫生学院,河北唐山 063200
2.齐鲁工业大学(山东省科学院) 生物研究所山东省科学院药物筛选技术重点实验室,山东济南 250103

收稿日期:2023-01-31 出版日期:2023-12-20 发布日期:2023-12-07
通信作者: * 靳梦(1985—),女,博士,研究员,硕士生导师,研究方向为神经系统疾病模型建立和神经药理学。E-mail:mjin1985@hotmail.com；张秀军(1966—),男,博士,教授,博士生导师,研究方向为药理学、毒理学。E-mail:zhangxiujun66@163.com
作者简介:时瑞碟(1997—),女,硕士研究生,研究方向为神经药理学。E-mail:shiruidie@163.com
基金资助:
济南市“新高校20条”项目(2021GXRC106);齐鲁工业大学(山东省科学院)科教产融合试点工程项目(2022PY033);齐鲁工业大学(山东省科学院)科教产融合试点工程项目(2022JBZ01-06)

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

SHI Ruidie¹^,²(), GAO Xin², WANG Baokun², GAO Daili², JIN Meng²^,^*(), ZHANG Xiujun¹^,^*()

1. College of Psychology and Mental Health, North China University of Science and Technology, Tangshan 063200, China
2. Key Laboratory of Drug Screening Technology of Shandong Academy of Sciences, Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, China

Received:2023-01-31 Online:2023-12-20 Published:2023-12-07

摘要/Abstract

摘要：

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,80 μmol/L六水合氯化铝模型对照组,80 μmol/L六水合氯化铝与6 μmol/L多奈哌齐阳性对照组,80 μmol/L六水合氯化铝与不同浓度(5、10、20 μmol/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。

关键词: 阿尔茨海默病, 六水合氯化铝, 自噬, 斑马鱼, 木蝴蝶苷A

Abstract:

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 (AlCl₃) was used. Wild-type zebrafish AB larvae at 3 dpf(days post fertilization) were divided into different groups, including negative control group, AlCl₃ (80 μmol/L) model control group, AlCl₃ (80 μmol/L) combined with donepezil (6 μmol/L) positive control group, and AlCl₃ (80 μmol/L) combined with different concentrations (5, 10, and 20 μmol/L) of oroxin A test group. At 6 dpf, zebrafish behavior was monitored and analyzed using zebrafish light-dark locomotion test. Aβ deposition 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 Aβ deposition and AchE activity of zebrafish induced by AlCl₃. The expression of autophagy-related genes tended to be normal after oroxin A treatment. This study preliminarily revealed that oroxin A alleviated AlCl₃-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.

Key words: Alzheimer's disease, aluminum chloride hexahydrate, autophagy, zebrafish, oroxin A

中图分类号:

R965

时瑞碟, 高鑫, 王宝堃, 高代丽, 靳梦, 张秀军. 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[J]. 山东科学, 2023, 36(6): 28-37.

SHI Ruidie, GAO Xin, WANG Baokun, GAO Daili, JIN Meng, ZHANG Xiujun. Anti-Alzheimer's disease activity of oroxin A and its mechanism of action based on zebrafish model[J]. Shandong Science, 2023, 36(6): 28-37.

图/表 6

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参考文献 22

[1]	TATULIAN S A. Challenges and hopes for Alzheimer's disease[J]. Drug Discovery Today, 2022, 27(4): 1027-1043. DOI: 10.1016/j.drudis.2022.01.016. pmid: 35121174
[2]	TAM K, JU Y J. Pathological mechanisms and therapeutic strategies for Alzheimer's disease[J]. Neural Regeneration Research, 2022, 17(3): 543. DOI: 10.4103/1673-5374.320970. pmid: 34380884
[3]	SRIRAM N, SAH M K. Regulatory insights into progression of cancer and Alzheimer's disorder from autophagy perspective[J]. Molecular Biology Reports, 2021, 48(12): 8227-8232. DOI: 10.1007/s11033-021-06838-4. pmid: 34665401
[4]	FANG E F, HOU Y J, PALIKARAS K, et al. Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer's disease[J]. Nature Neuroscience, 2019, 22(3): 401-412. DOI: 10.1038/s41593-018-0332-9. pmid: 30742114
[5]	SUN C, ZHANG SS, BA S K, et al. Eucommia ulmoides olive male flower extracts Ameliorate alzheimer's disease-like pathology in zebrafish via regulating autophagy, acetylcholinesterase, and the dopamine transporter[J]. Frontiers in Molecular Neuroscience, 2022, 15: 901953. DOI: 10.3389/fnmol.2022.901953.
[6]	国家药典委员会. 中华人民共和国药典2020年版二部[M]. 北京: 中国医药科技出版社, 2020.
[7]	CHENG X D, HUANG T T, WANG C H, et al. Natural compound library screening identifies oroxin A for the treatment of myocardial ischemia/reperfusion injury[J]. Frontiers in Pharmacology, 2022, 13: 894899. DOI: 10.3389/fphar.2022.894899.
[8]	鲁冰冰. 木蝴蝶苷A对食管鳞癌增殖的抑制作用及其机制的研究[D]. 郑州: 郑州大学, 2020.
[9]	任擎宇, 靳梦, 商学良, 等. 基于网络药理学和斑马鱼模型的金盏菊缓解阿尔茨海默病症状及作用机制[J]. 中国药理学通报, 2022, 38(3): 429-436. DOI: 10.12360/CPB202108075.
[10]	BASNET R M, ZIZIOLI D, TAWEEDET S, et al. Zebrafish larvae as a behavioral model in neuropharmacology[J]. Biomedicines, 2019, 7(1): 23. DOI: 10.3390/biomedicines7010023.
[11]	SHENOY A, BANERJEE M, UPADHYA A, et al. The brilliance of the zebrafish model: perception on behavior and alzheimer's disease[J]. Frontiers in Behavioral Neuroscience, 2022, 16: 861155. DOI: 10.3389/fnbeh.2022.861155.
[12]	PATIL D N, PATIL S A, SISTLA S, et al. Comparative biophysical characterization: A screening tool for acetylcholinesterase inhibitors[J]. PLoS One, 2019, 14(5): e0215291. DOI: 10.1371/journal.pone.0215291.
[13]	SAXENA M, DUBEY R. Target enzyme in Alzheimer's disease: acetylcholinesterase inhibitors[J]. Current Topics in Medicinal Chemistry, 2019, 19(4): 264-275. DOI: 10.2174/1568026619666190128125912. pmid: 30706815
[14]	SENGER M R, SEIBT K J, GHISLENI G C, et al. Aluminum exposure alters behavioral parameters and increases acetylcholinesterase activity in zebrafish (Danio rerio) brain[J]. Cell Biology and Toxicology, 2011, 27(3): 199-205. DOI: 10.1007/s10565-011-9181-y. pmid: 21240652
[15]	WALSH D M, SELKOE D J. Amyloid β-protein and beyond: the path forward in Alzheimer's disease[J]. Current Opinion in Neurobiology, 2020, 61: 116-124. DOI: 10.1016/j.conb.2020.02.003. pmid: 32197217
[16]	BUSCHE M A, WEGMANN S, DUJARDIN S, et al. Tau impairs neural circuits, dominating amyloid-β effects, in Alzheimer models in vivo[J]. Nature Neuroscience, 2019, 22(1): 57-64. DOI: 10.1038/s41593-018-0289-8. pmid: 30559471
[17]	DENG Z Q, DONG Y, ZHOU X T, et al. Pharmacological modulation of autophagy for Alzheimer's disease therapy: Opportunities and obstacles[J]. Acta Pharmaceutica Sinica B, 2022, 12(4): 1688-1706. DOI: 10.1016/j.apsb.2021.12.009. pmid: 35847516
[18]	PICKFORD F, MASLIAH E, BRITSCHGI M, et al. The autophagy-related protein beclin1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice[J]. The Journal of Clinical Investigation, 2008, 118(6): 2190-2199. DOI: 10.1172/JCI33585.
[19]	JAEGER P A, PICKFORD F, SUN C H, et al. Regulation of amyloid precursor protein processing by the beclin1 complex[J]. PLoS One, 2010, 5(6): e11102. DOI: 10.1371/journal.pone.0011102.
[20]	MCALPINE F, WILLIAMSON L E, TOOZE S A, et al. Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2[J]. Autophagy, 2013, 9(3): 361-373. DOI: 10.4161/auto.23066. pmid: 23291478
[21]	任李梅, 杨松, 卢姝言, 等. 人参总皂苷在巨噬细胞RAW264.7中降低脂多糖诱导的炎症和氧化应激[J]. 中国医院药学杂志, 2022, 42(9): 896-901. DOI: 10.13286/j.1001-5213.2022.09.04.
[22]	CARVALHO C, SANTOS M S, OLIVEIRA C R, et al. Alzheimer's disease and type 2 diabetes-related alterations in brain mitochondria, autophagy and synaptic markers[J]. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2015, 1852(8): 1665-1675. DOI: 10.1016/j.bbadis.2015.05.001.

基因名称	引物序列
rpl13a	上游: TCTGGAGGACTGTAAGAGGTATGC
	下游: AGACGCACAATCTTGAGAGCAG
beclin1	上游: GTTCAGGTGGTCTGCGTTTT
	下游: GCAAACAGAAGCCAGTGTCA
ulk1b	上游: AGGCCGAAAGTCTCACTTCA
	下游: AGCCATGTACATCGGAGACC
ulk2	上游: ACCTCTGATTGGCTGACAAAAT
	下游: GAGATTGCAAGAGGCTTGAGTT
atg7	上游: AGAGTCCAGTCCGATGTC
	下游: A GAAGTAACAGCCGAGACG

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

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