兰州大学机构库 >药学院
利用秀丽隐杆线虫模型研究甘松中单体化合物ethyl caffeate抗AD的作用机制
Alternative TitleStudy on the anti-AD mechanism of ethyl caffeate from Nardostachys jatamansi using Caenorhabditis elegans models
白雪
Subtype硕士
Thesis Advisor张占欣
2023-05-28
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline药学
Keyword阿尔茨海默病 Alzheimer's disease 秀丽隐杆线虫 Caenorhabditis elegans ethyl caffeate ethyl caffeate 胰岛素/胰岛素样生长因子-1信号通路 insulin/insulin-like growth factor-1 signalling pathway 蛋白平衡降解体系 protein homeostasis degradation system
Abstract

阿尔茨海默病(Alzheimer's disease,AD)是一种与衰老相关的神经退行性疾病,具有起病隐匿、病因复杂和诊断困难等特点。随着发病率的不断增长,AD现已成为严重威胁我国居民健康的重大疾病。目前美国食品药品监督管理局(the US Food and Drug Administration,FDA)批准的用于治疗AD的药物十分有限。这些药物只针对轻到中度的AD患者,只能短暂缓解症状,并不能阻止疾病进程,并且副作用明显,所以寻找新的更加安全有效的抗AD药物十分必要。

越来越多的证据表明,β-淀粉样蛋白(amyloid-β peptides,Aβ)、氧化应激和神经炎症是AD进程中的关键促因。有文献报道,中药甘松(Nardostachys jatamansi (D.Don) DC.)乙醇提取物和乙酸乙酯提取物具有抗神经炎症、抗氧化和神经保护等多种药理活性,这提示甘松中可能含有多种抗AD活性成分。本文首先选用转基因秀丽隐杆线虫CL4176 AD病理模型,对从甘松中分离得到的14个天然单体化合物进行了抗AD活性评估,结果表明有7个倍半萜类成分(化合物1-5、7、8)、1个酚类成分(化合物12)和一个苷类成分(化合物13)具有潜在抗AD活性。其中,优选化合物12(ethyl caffeate)能以剂量依赖性的方式显著延缓CL4176线虫的麻痹进程,同时还能显著缓解CL2355线虫对外源性5-羟色胺(5-hydroxytryptamine,5-HT)超敏的麻痹样症状。这表明ethyl caffeate不仅能抑制Aβ1-42诱导的体壁肌肉细胞毒性,还能抑制Aβ1-42诱导的神经细胞毒性,说明其具有良好的、潜在的抗AD活性。故本文利用秀丽隐杆线虫模式生物进一步探讨了ethyl caffeate体内发挥抗AD作用的分子机制。

硫黄素S(thioflavine S,ThS)染色结果表明ethyl caffeate显著降低了CL2006线虫头部的Aβ沉积斑;Western blot实验结果表明ethyl caffeate显著抑制了Aβ单体和毒性寡聚体的蛋白表达水平;Aβ mRNA表达水平检测结果表明空白对照组和药物处理组之间没有显著性差异,这提示ethyl caffeate介导的Aβ沉积斑的降低可能是调节Aβ转录后水平所致。利用CL2179线虫进一步研究发现ethyl caffeate对外源蛋白Aβ1-42的抑制作用呈现特异性。氧化应激在AD进程中也发挥着重要作用,ethyl caffeate能将百草枯诱导的高活性氧(reactive oxygen species,ROS)水平降至接近正常状态,这可能与其降低Aβ沉积斑有关。胰岛素/胰岛素样生长因子-1信号(insulin/insulin-like growth factor-1 signaling,IIS)通路在增强线虫抗应激和抑制Aβ毒性方面起着重要作用,利用实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)技术对该通路中与抗应激和抗胁迫相关的基因进行了检测,结果表明ethyl caffeate显著上调了daf-16、skn-1、hsf-1、sod-3、gst-4和hsp-16.2基因的表达。利用荧光显微技术研究发现,ethyl caffeate显著促进了转录因子DAF-16和SKN-1的核转位,同时增强了下游靶标SOD-3、GST-4和HSP-16.2的表达,这提示ethyl caffeate可能部分通过IIS通路抑制Aβ沉积斑累积,发挥抗AD作用。Aβ聚集会打破机体蛋白质的动态平衡,蛋白平衡降解体系在清除错误折叠或异常积聚Aβ方面至关重要。实验结果表明,ethyl caffeate显著增强了与内质网未折叠蛋白反应(endoplasmic reticulum unfolded protein reaction,UPRER)及线粒体未折叠蛋白反应(mitochondrial unfolded protein response,UPRmt)相关的分子伴侣HSP-4、HSP-6和HSP-60的表达,同时还增强了蛋白酶体亚基RPT-3的表达,这说明ethyl caffeate可能部分通过分子伴侣途径和泛素-蛋白酶体途径降解和清除未折叠、错误折叠或异常积聚的Aβ蛋白,发挥抗AD作用。此外,ethyl caffeate还降低了聚谷氨酰胺(polyglutamine,polyQ)蛋白的异常积聚水平,提示其对基于蛋白质异常积聚或错误折叠的其它神经退行性疾病可能也具有治疗潜力。

本研究首次使用秀丽隐杆线虫模型,主要探讨了ethyl caffeate发挥抗AD作用的分子机制。本研究的结果将为甘松及其所含天然化合物ethyl caffeate的高值化利用与开发新型抗AD先导化合物提供更多的理论支持。

 

Other Abstract

Alzheimer's disease (AD) is an aging-related neurodegenerative disease characterized by insidious onset, complex etiology and difficulty in diagnosis. With the increasing incidence of AD, it has become a serious threat to the health of the population in China. At present, the US Food and Drug Administration (FDA) has approved very limited drugs for the treatment of AD. These drugs are only for patients with mild to moderate AD, which can only relieve symptoms for a short period of time, but not stop the progression of the disease, as well as having significant side-effects, so there is a need to find safer and more effective anti-AD drugs.

There are growing evidences that amyloid-β peptides (Aβ), oxidative stress and neuroinflammation are key contributors to the AD process. It has been reported that the ethanolic and ethyl acetate extracts of Nardostachys jatamansi (D.Don) DC. showed various pharmaceutical activities including anti-inflammatory, antioxidant and neuroprotective activities, suggesting that it may contain various anti-AD active components. In this study, the anti-AD activity of 14 natural compounds isolated from Nardostachys jatamansi was evaluated using a transgenic Caenorhabditis elegans CL4176 AD pathological model. The results showed that there were seven sesquiterpene components (compounds 1-5, 7, 8), one phenolic component (compound 12) and one glycoside component (compound 13) possessing potential anti-AD activity. Of these, the preferred compound 12 (ethyl caffeate) significantly delayed the paralysis of CL4176 nematodes in a dose-dependent manner, while also significantly alleviating the paralysis-like symptoms of CL2355 hypersensitivity to exogenous 5-hydroxytryptamine (5-HT). This indicated that ethyl caffeate could not only alleviate Aβ1-42-induced muscle cytotoxicity in the body wall, but also could inhibit Aβ1-42-induced neurotoxicity, indicating it had a good and potential anti-AD activity. Therefore, this study further explored the molecular mechanism of its anti-AD effect in vivo using C. elegans models.

Thioflavine S (ThS) staining showed that ethyl caffeate significantly reduced Aβ deposition spots on the head of CL2006 nematodes and the results of Western blot showed that ethyl caffeate significantly inhibited the protein expression of Aβ monomers and toxic oligomers, while Aβ mRNA expression levels were not significantly different between the blank control and drug-treated groups, thus suggesting that the reduction of Aβ deposition spots mediated by ethyl caffeate may be due to the regulation of Aβ post-transcriptional levels. Using CL2179 nematodes, further studies revealed that ethyl caffeate showed a specific inhibitory effect on the exogenous protein Aβ1-42. Oxidative stress also plays an important role in the process of AD, ethyl caffeate reduced paraquat-induced high levels of reactive oxygen species (ROS) to near normal, which may be related to its reduction of Aβ deposition spots. The insulin/insulin-like growth factor-1 signaling (IIS) pathway plays an important role in the enhancement of the resistance to stress and inhibition of Aβ toxicity in nematodes. Using quantitative real-time polymerase chain reaction (qRT-PCR), we examined genes related to stress resistance in this pathway and the results showed that ethyl caffeate significantly upregulated the expression of daf-16, skn-1, hsf-1, sod-3, gst-4 and hsp-16.2 genes. By using fluorescence microscopy, ethyl caffeate significantly promoted the nuclear translocation of transcription factors DAF-16 and SKN-1, and enhanced the expression of downstream targets SOD-3, GST-4 and HSP-16.2, indicating that ethyl caffeate may exert its anti-AD effect in part through the IIS pathway to inhibit the accumulation of Aβ deposits. Aβ accumulation can disrupt the dynamic equilibrium of proteins in the body, so protein homeostatic degradation systems are essential in removing misfolded or abnormally deposited Aβ. The results showed that ethyl caffeate significantly enhanced the expression of the molecular chaperones HSP-4, HSP-6 and HSP-60, which are associated with the endoplasmic reticulum unfolded protein reaction (UPRER) and mitochondrial unfolded protein response (UPRmt). It also enhanced the expression of the proteasome subunit RPT-3. These suggested that ethyl caffeate may work in part through the molecular chaperone pathway and the ubiquitin-proteasome pathway to degrade and remove unfolded, misfolded or abnormally accumulated Aβ proteins to exert anti-AD effects. In addition, ethyl caffeate decreased the level of abnormal accumulation of polyglutamine (polyQ) protein, suggesting that it may also have therapeutic potential for other neurodegenerative diseases based on the abnormal accumulation or misfolding of proteins.

This study is the first time to use the C. elegans models to investigate the molecular mechanism of the anti-AD effect of ethyl caffeate. The results of this study will provide more theoretical support for the high value use of Nardostachys jatamansi and its natural compound ethyl caffeate as well as the development of new anti-AD lead compounds.

MOST Discipline Catalogue医学 - 药学(可授医学、理学学位)
URL查看原文
Language中文
Other Code262010_220200941540
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/537005
Collection药学院
Affiliation
兰州大学药学院
Recommended Citation
GB/T 7714
白雪. 利用秀丽隐杆线虫模型研究甘松中单体化合物ethyl caffeate抗AD的作用机制[D]. 兰州. 兰州大学,2023.
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