石菖蒲单体对β-淀粉样蛋白致神经元损伤模型的保护作用研究

发布时间：2018-06-23 10:46

本文选题：β-淀粉样蛋白 + β-细辛醚　；参考：《广东药学院》2012年硕士论文

【摘要】：研究背景：阿尔兹海默症(Alzheimer's Disease,AD)是一种最为常见的中枢神经系统退行性疾病,为痴呆的最主要类型。目前“Aβ假说”被广泛地接受为AD最主要的发病机理,即老年斑的主要组成成分——β-淀粉样蛋白(amyloid-β,AB)是AD发病进程的中心机制,其在特定脑区内聚集,引发相应的神经毒性作用,造成突触损伤,神经元死亡,从而导致了AD患者记忆衰退和认知功能障碍等相应症状的产生。目前,AD的治疗研究策略主要包括三个方面：一是开发新的低毒性的拟胆碱药物；二是神经营养物质的应用,包括直接脑室灌注神经营养因子和神经营养因子基因修饰细胞的脑内移植治疗；三是干细胞的移植治疗。但这些疗法基本在于减慢神经元的退变或加速神经元的产生,并没有从根本上阻止AD患者的病变,其远期治疗前景不容乐观。而中医药临床表明,以石菖蒲为主的药方是治疗AD方剂的基本结构,位居单味药使用频次第1位,疗效肯定。但石菖蒲抗AD的药效部位及抗AD的“Aβ”机制尚未阐明,影响了石菖蒲抗AD疗效的进一步提高。我室前期工作证明,石菖蒲挥发油部位对AD模型小鼠学习记忆能力有明显提升作用,作用机制可能与其拮抗凋亡相关因子表达有关,提示挥发油部位有可能用于AD的防治。为进一步探讨石菖蒲挥发油部位抗AD及其发挥神经保护作用的有效成分,为石菖蒲抗AD及用于神经保护药物的研发提供科学依据,本实验首先用Aβ1-42寡聚体对体外培养的原代皮层神经元进行优化筛选,建立了Aβ1-42寡聚体致神经元损伤模型。在此模型上探讨β-细辛醚(β-asarone)、丁香酚(Eugenol)以及β-细辛醚联合丁香酚共孵育对Aβ1-42致神经元损伤模型的保护作用及机制,为石菖蒲抗AD及用于神经保护药物的研发提供科学依据。研究目的： 1.观察β-细辛醚和丁香酚对正常状态下原代皮层神经元和PC12细胞的形态和细胞活力的影响,初步了解β-细辛醚和丁香酚的神经保护作用； 2.观察p-细辛醚、丁香酚及p-细辛醚联合丁香酚共孵育对Aβ1-42寡聚体诱导的原代皮层神经元和PC12细胞损伤模型的保护作用,并探讨其作用机理。研究方法：实验一 1.首先取胎鼠的皮层组织进行原代皮层神经元的培养,培养第六天进行免疫细胞化学染色鉴定神经元； 2. Aβ1-42寡聚体诱导的皮层神经元损伤模型的建立,ELISA检测相关因子的释放水平；3.观察β-细辛醚和丁香酚对皮层神经元的细胞活力的影响； 4.原代皮层神经元培养第六天加入Aβ1-42寡聚体建立细胞模型,24小时后再用β-细辛醚和丁香酚作用于细胞。三天后提取蛋白进行凋亡相关因子的检测；实验二1.石菖蒲主要有效成分β-细辛醚和丁香酚对正常状态下PC12细胞形态和细胞活力的影响； 2.建立PC12细胞的Aβ1-42寡聚体诱导损伤模型； 3.PC12细胞培养第六天加入Aβ1-42寡聚体建立细胞模型,24小时后再用p-细辛醚和丁香酚作用于细胞。三天后提取RNA进行凋亡相关基因的检测； 4.通过Tunel法探讨p-细辛醚和丁香酚对Aβ1-42寡聚体诱导的PC12细胞的保护作用机理。研究结果：实验一 1.培养至第六天的原代皮层神经元胞体的突起明显,免疫细胞化学染色显示,神经元标志物呈阳性； 2.通过ELISA检测TNF-α的释放水平,建立起Aβ1-42寡聚体诱导的皮层神经元损伤模型； 3.培养至第三天的皮层神经元分别与10-10-10-8Mp-细辛醚、丁香酚以及p-细辛醚联合丁香酚共孵育24h,与正常培养的神经元相比,10-6β-细辛醚和10-7M丁香酚组细胞存活率最高； 4.Western blotting技术检测凋亡相关蛋白,显示Aβ1-42寡聚体作用于皮层神经元6h后,促凋亡蛋白Bax表达开始增加,至24h达到较高水平；与之相反,抗凋亡蛋白Bcl-2表达随Aβ1-42寡聚体作用时间延长逐渐降低； 5.10-10-10-8Mp-细辛醚和丁香酚可明显拮抗Aβ1-42寡聚体引起的促凋亡蛋白Bax表达上调以及抗凋亡蛋白Bcl-2表达下降。实验二1.培养至第三天的PC12细胞与10-10-10-8Mβ-细辛醚和丁香酚分别共孵育24h,与正常培养的PC12细胞相比,10-6β-细辛醚和10-7M丁香酚组细胞存活率最高； 2.RT-PCR技术检测凋亡相关基因,显示Aβ1-42寡聚体作用于PC12细胞后,促凋亡蛋白Bax表达开始增加,抗凋亡蛋白Bcl-2表达逐渐降低； 3.10-10-10-8Mp-细辛醚和丁香酚可明显拮抗Aβ1-42寡聚体引起的PC12细胞促凋亡基因Bax表达上调以及抗凋亡基因Bcl-2表达下降； 4.TUNEL方法检测PC12细胞凋亡：β-细辛醚、丁香酚及其混合物的预孵育组,与Aβ1-42寡聚体对照组相比较,阳性细胞数量明显减少。p-细辛醚联合丁香酚用药组优于单体的单独作用组。结论： 1.Aβ1-42寡聚体能显著抑制神经元的再生,这种毒性效应可能与其促进凋亡相关因子的表达有关。 2.β-细辛醚和丁香酚能明显拮抗Aβ1-42寡聚体引发的神经元(PC12细胞)细胞凋亡,这可能与其能抑制凋亡相关因子的表达有关。 3.对于Aβ1-42寡聚体诱导的原代皮层神经元和PC12细胞损伤模型的保护作用,β-细辛醚联合丁香酚组明显优于单体的单独作用组。这为石菖蒲用于抗老年性痴呆药物的研发提供了科学的依据。
[Abstract]:Research background:
Alzheimer's Disease (AD) is the most common degenerative disease of the central nervous system and is the most important type of dementia. Now the "A beta hypothesis" is widely accepted as the main pathogenesis of AD, that is, the main component of the senile plaque, beta amyloid (amyloid- beta, AB), is the central machine for the pathogenesis of AD. In a specific brain area, it is aggregated in a specific brain area, triggering a corresponding neurotoxic effect, causing synaptic damage and causing death of neurons, resulting in the resulting symptoms of memory decline and cognitive impairment in AD patients.
At present, the treatment research strategy of AD mainly includes three aspects: first, the development of new low toxic quasi choline drugs; two is the use of neurotrophic substances, including direct ventricle perfusion neurotrophic factor and neurotrophic factor gene modified cell transplantation in the brain; three is stem cell transplantation treatment. But these treatments are basically To slow down the degeneration of neurons or accelerate the production of neurons, it does not fundamentally prevent the disease of AD patients. The prospect of long-term treatment is not optimistic. The clinical manifestation of traditional Chinese medicine indicates that the prescription of Acorus calamus is the basic structure for the treatment of AD prescription, which is the first place of the single drug use frequency, but the effect of Acorus calamus on AD The mechanism of "A beta" against AD has not been clarified, which has affected the further improvement of the efficacy of Acorus calamus against AD.
The early work of my room has proved that the volatile oil parts of Acorus calamus have a significant effect on the learning and memory ability of AD model mice. The mechanism may be related to its antagonism to the expression of apoptosis related factors, suggesting that the part of the volatile oil may be used for the prevention and control of AD. To further discuss the anti AD and the neuroprotective effect of the volatile oil from the calamus The effective components, which provide a scientific basis for the anti AD of Acorus calamus and the research and development of neuroprotective drugs, first optimized the primary cultured cortical neurons in vitro by A beta 1-42 oligomer, and established the neuron damage model of A beta 1-42 oligomers. Asarone
The protective effect and mechanism of CO incubation with A beta 1-42 induced neuron damage model can provide a scientific basis for the anti AD of Acorus calamus and the research and development of neuroprotective drugs.
The purpose of the study is:
1. the effects of beta asarone and eugenol on the morphology and cell viability of primary cortical neurons and PC12 cells in normal state were observed, and the neuroprotective effects of beta asarone and eugenol were preliminarily understood.
2. the protective effect of p- asarone, eugenol and p- asarone combined with eugenol on the primary cortical neurons and PC12 cell damage models induced by A beta 1-42 oligomers was observed and the mechanism of its action was discussed.
Research methods:
Experiment 1
1. first, the cortical tissues of fetal rats were cultured in primary cortical neurons for sixth days to identify neurons by immunocytochemical staining.
2. A beta 1-42 oligomer induced cortical neuron damage model was established, and ELISA was used to detect the release level of related factors; 3. the effects of beta asarone and eugenol on the cell viability of cortical neurons were observed.
4. the primary cultured cortical neurons were cultured for sixth days by adding A beta 1-42 oligomer to establish cell model. After 24 hours, beta asarone and eugenol were used to act on the cells. After three days, the protein was detected for the detection of apoptosis related factors.
Experiment two. The effects of 1. asarone and eugenol on the morphology and cell viability of PC12 cells under normal conditions.
2. to establish PC12 cell induced A beta 1-42 oligomer induced injury model.
3.PC12 cells were cultured for sixth days to add A beta 1-42 oligomer to establish cell model. After 24 hours, p- asarone and eugenol were used to act on the cells. After three days, RNA was detected for the detection of apoptosis related genes.
4. to explore the protective mechanism of p- asarone and eugenol on PC12 cells induced by A beta 1-42 oligomers by Tunel.
The results of the study:
Experiment 1
1. the neurites of the primary cortical neurons cultured for up to sixth days were obvious. Immunocytochemical staining showed that the neurons were positive.
2. to detect the release level of TNF- alpha by ELISA, and establish a cortical neuron injury model induced by A beta 1-42 oligomers.
3. the cortical neurons cultured for third days incubated 24h with 10-10-10-8Mp- asarone, eugenol and p- asarone combined with eugenol. Compared with the normal cultured neurons, the survival rate of 10-6 beta asarine and 10-7M eugenol group was the highest.
4.Western blotting technique detected apoptosis related proteins, which showed that after A beta 1-42 oligomer acted on 6h of cortical neurons, the expression of apoptotic protein Bax began to increase and reached a higher level to 24h. On the contrary, the expression of anti apoptotic protein Bcl-2 gradually decreased with the prolongation of the action time of A beta 1-42 oligomer.
5.10-10-10-8Mp- asarone and eugenol can significantly antagonize upregulated expression of Pro apoptotic protein Bax and decrease the expression of anti apoptotic protein Bcl-2 in A beta 1-42 oligomers.
Experiment two 1. PC12 cells cultured to third days were incubated with 10-10-10-8M beta asarone and eugenol respectively for 24h. Compared with normal cultured PC12 cells, the survival rate of 10-6 beta asarone and 10-7M eugenol group was the highest.
Apoptosis related genes were detected by 2.RT-PCR technology, which showed that A beta 1-42 oligomer acted on PC12 cells, and the expression of apoptotic protein Bax began to increase, and the expression of anti apoptotic protein Bcl-2 decreased gradually.
3.10-10-10-8Mp- asarone and eugenol could obviously antagonize the up regulation of Bax expression of PC12 cell apoptosis gene Bax and the decrease of Bcl-2 expression of anti apoptotic gene induced by A beta 1-42 oligomer.
4.TUNEL method was used to detect the apoptosis of PC12 cells: pre incubating group of beta asarone, eugenol and its mixture, compared with A beta 1-42 oligomer control group, the number of positive cells significantly reduced the group of.P- asarone combined with eugenol group better than the single body.
Conclusion:
1.A beta 1-42 oligomers can significantly inhibit the regeneration of neurons. This toxic effect may be related to the expression of apoptosis related factors.
2. beta asarone and eugenol can obviously antagonize the apoptosis of neurons (PC12 cells) induced by A beta 1-42 oligomers, which may be related to the inhibition of the expression of apoptosis related factors.
3. for the protective effect of A beta 1-42 oligomer induced primary cortical neurons and PC12 cell damage models, beta asarone combined with eugenol group is obviously better than the single action group. This provides a scientific basis for the development of Acorus calamus for the development of anti Alzheimer's drugs.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R749.16

【共引文献】