薰衣草益智药效组分及基于抗氧化作用的机制研究

发布时间：2017-12-28 00:16

  本文关键词：薰衣草益智药效组分及基于抗氧化作用的机制研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

  更多相关文章： 薰衣草 精油 芳樟醇 阿尔兹海默病 学习记忆 抗氧化 神经毒性

【摘要】：阿尔兹海默症(Alzheimer's Disease,AD)是最常见的一种神经退行性疾病,可导致认知障碍、人格改变、行为异常等症状。氧化应激与AD病理进程密切相关,AD中Aβ的沉积与氧化应激相互诱导,导致线粒体功能损伤,诱发神经细胞凋亡。同时核因子 E2 相关因子 2(Nuclear factor-erythroid 2-related factor 2,Nrf2)-血红素加氧酶-1(hemeoxygenase-1,HO-1)通路在机体抗氧化中发挥重要作用。所以,近年来氧化应激所致损伤已置于AD研究的重要位置,并将从传统天然药物中寻找研发抗氧化活性物质作为开发AD防治药物的新途径。薰衣草(Lavandula angustifolia Mill.)作为一种传统中药,能有效治疗焦虑、抑郁、失眠、疼痛、惊厥、脑血栓等神经系统疾病。薰衣草精油(Lavender oil,LO)及薰衣草水提物(Lavender extract,LE)都报道有中枢神经系统活性,并表现出明显的抗氧化作用。研究表明,主要成分(-)-芳樟醇(Linalool,LI)能通过调控Nrf2-HO-1通路发挥抗氧化、抗炎等作用。因此,我们推测薰衣草对AD学习记忆损伤有改善潜力。本论文拟通过多种体内外模型对薰衣草的不同益智成分进行学习记忆改善作用评价,并从抗氧化入手探索相关的作用机制;此外,为了保证薰衣草用药时的神经系统安全性,本课题还拟研究薰衣草精油对正常动物神经行为,尤其是运动行为的影响,并对其相关作用机制进行探索,为将薰衣草开发为AD防治药物提供有效性、安全性的实验基础和依据。主要研究内容如下:1.薰衣草益智活性成分的筛选及认知改善作用评价首先对LO与LE神经保护及益智作用进行初步的评价及比较。通过H202、Aβ25-35诱导的PC12细胞损伤模型和东莨菪碱诱导认知损伤小鼠模型,对LO和LE的神经活性进行初步评价。结果表明LO有明显的神经细胞损伤保护作用,并改善东莨菪碱导致的认知障碍;而LE的体内外药效作用均不明显,所以选择LO做进一步药效研究。先建立GC-MS方法对薰衣草精油进行成分确定和含量分析,发现其主要成分为芳樟醇(Linalool,LI)约为37.96%。接着同时对LO及LI的益智作用进行研究,建立Aβ1-40海马注射和D-galactose(D-gal)联合AlC13诱导AD小鼠模型,水迷宫及避暗结果显示,LO与LI都表现出明显改善AD模型中学习记忆损伤的作用。2.薰衣草精油及芳樟醇基于抗氧化的神经保护机制研究对于体内模型,取Aβ1-40所致AD模型的海马组织进行生物化学、Western blot、染色分析发现,LO或LI可以逆转模型中抗氧化相关酶SOD和GPX活性的降低,和MDA的升高;并抑制caspase-9、caspase-3的激活,恢复MMP水平,使凋亡率明显下降。同时,在D-gal联合AlCl3诱导模型海马中,LO或LI同样表现了抗氧化的作用,并且提高神经可塑性相关蛋白CaMKII,p-CaMKII,BDNF和TrkB的表达。此外,对于H2O2、Aβ25-35诱导的体外模型,LI可以逆转ROS、NO的水平、caspase-9、caspase-3活性、MMP和细胞凋亡率。所以,LO与LI在神经保护中表现出明显的抗氧化、抗凋亡的作用。进一步对Nrf2-HO-1通路在LI抗氧化神经保护中的作用进行研究。将LI对PC12细胞进行直接干预,Western blot结果表明:LI可以时间依赖和浓度依赖地促进HO-1、Nrf2的表达及Akt的激活,并且促进Nrf2由胞质到胞内的转移;当使用PI3K/Akt抑制剂LY249002或HO-1的抑制剂ZnPP对细胞进行预处理后,LI所诱导的HO高表达受到抑制,并且LI对抗氧化的保护作用也被破坏。此外,LI也使Aβ1-40和D-gal+AlCl3诱导小鼠海马中HO-1、Nrf2的表达升高。可以推断,LI对神经细胞抗氧化保护作用可能是通过PI3K/Akt-Nrf2-HO-1通路介导。3.薰衣草精油对正常小鼠神经行为的影响先通过一系列行为学实验观察到LO对正常大鼠的运动能力有明显的抑制作用,对情绪和学习记忆能力影响不显著。进而设置LO单次给药或多次给药模式,研究小鼠运动特征随给药剂量和时间因素的变化趋势,结果表明LO使正常小鼠运动速度减慢,时长增加,对其运动有抑制作用;但并不影响小鼠的运动协调性,表现出改善促进作用。并且LO的作用受给药剂量的影响较为显著,在低剂量(0.35 g/kg)时LO不改变正常的运动功能,证明了 LO作为益智药物应用的安全性。最后通过神经递质、受体表达分析,推测运动的变化与脑内多巴胺能神经系统活性及5-HT水平相关。综上所述,本课题首次从益智作用、作用机制、“神经毒性”等多个方面对薰衣草进行了较全面系统的研究,结果表明LO有明显的益智作用,而芳樟醇为精油中的主要活性成分;这种益智作用的机制与抗氧化、抗凋亡的活性相关,并受PI3K/Akt-Nrf2-HO-1通路的调控;此外,LO引起正常小鼠运动功能变化的剂量显著地高于益智药效剂量,说明了 LO作为益智药物使用的安全性。本课题的研究将为从薰衣草中开发AD潜在预防治疗药物提供更多的实验基础。
[Abstract]:Alzheimer's Disease (AD) is the most common neurodegenerative disease, which can cause symptoms such as cognitive impairment, personality change, and abnormal behavior. Oxidative stress is closely related to the pathological process of AD. The deposition of A beta in AD and oxidative stress induce each other, causing mitochondrial dysfunction and inducing neuronal apoptosis. Meanwhile, the nuclear factor E2 related factor 2 (Nuclear factor-erythroid 2-related factor 2, Nrf2) - heme oxygenase -1 (hemeoxygenase-1, HO-1) pathway plays an important role in the body's antioxidant capacity. Therefore, in recent years, oxidative stress injury has been placed in the important position of AD research. It will seek new antioxidants from traditional natural medicines as a new way to develop AD drugs. Lavandula angustifolia Mill., as a traditional Chinese medicine, can effectively treat anxiety, depression, insomnia, pain, convulsion, cerebral thrombosis and other neurological diseases. Lavender oil (LO) and Lavender extract (LE) are reported to have central nervous system activity and show obvious antioxidant effects. Research shows that the main component of (-) - linalool (Linalool, LI) can exert antioxidant and anti-inflammatory effect by regulating Nrf2-HO-1 pathway. Therefore, we speculate that lavender has the potential to improve the learning and memory impairment of AD. This paper intends to improve the learning and memory function evaluation through a variety of in vitro and in vivo models of different educational component of the lavender, and from the start to explore the mechanism of antioxidant related; in addition, in order to ensure the safety of the nervous system when using lavender, this paper also intends to study the lavender essential oil on normal animal nerve behavior, especially the effects of exercise behavior, and the related mechanisms were explored, to provide experimental basis for the development of lavender and on the basis of the effectiveness and safety of drugs for prevention and treatment of AD. The main research contents are as follows: 1.. Screening and cognitive improvement of lavender active ingredients. First, we evaluated and compared the effects of LO and LE on neuroprotection and intelligence. The neuroactivity of LO and LE was evaluated by H202, A beta 25-35 induced PC12 cell damage model and scopolamine induced mouse model of cognitive impairment. The results showed that LO had obvious protective effects on nerve cell injury and improved cognitive impairment induced by scopolamine, while the pharmacodynamic effects of LE were not obvious in vivo and in vitro, so LO was selected for further pharmacodynamic studies. We established the GC-MS method to analyze the components and determine the content of lavender essential oil, found that the main component of linalool (Linalool, LI) is about 37.96%. At the same time, we studied the intelligence function of LO and LI, and established A AD 1-40 hippocampus injection and D-galactose (D-gal) combined AlC13 induced AD mouse model. The water maze and dark avoidance results showed that LO and LI all significantly improved learning and memory impairment in AD model. 2. lavender essential oil and linalool study of neural protection mechanism based on the antioxidation in vivo model, A beta induced by 1-40 AD model in hippocampus of Biochemistry, Western and blot staining analysis found that LO or LI can reverse the antioxidant related enzymes in model SOD and GPX activity decreased, and MDA increased; and inhibit the activation of caspase-9 Caspase-3, the recovery level of MMP, the apoptosis rate decreased significantly. At the same time, in the hippocampus of D-gal combined with AlCl3, LO or LI also showed the antioxidant effect, and increased the expression of CaMKII, p-CaMKII, BDNF and TrkB of neural plasticity related protein. In addition, LI could reverse the level of ROS, NO, caspase-9, caspase-3 activity, MMP and apoptosis rate of H2O2 and A beta 25-35 induced in vitro model. Therefore, LO and LI show obvious antioxidation and anti apoptotic effects in neuroprotection. The role of Nrf2-HO-1 pathway in the protection of LI antioxidant nerve was further studied. LI direct intervention on PC12 cells, Western blot results show that LI can promote the time dependent and concentration dependent activation and the expression of Akt HO-1 and Nrf2, and promote Nrf2 transfer from cytoplasm to cytoplasm; when using the PI3K/Akt inhibitor LY249002 or HO-1 inhibitor ZnPP pretreatment on cells after high expression LI induced by HO was inhibited, and the protective effect of LI against oxidative destruction. In addition, LI also increased the expression of HO-1 and Nrf2 in the hippocampus of mice induced by A beta 1-40 and D-gal+AlCl3. It is inferred that the protective effect of LI on nerve cells may be mediated by the PI3K/Akt-Nrf2-HO-1 pathway. 3., the influence of lavender essential oil on the neurobehavior of normal mice was observed through a series of behavioral experiments. LO had a significant inhibitory effect on the motor ability of normal rats, but had no significant effect on mood and learning and memory abilities. And then set the LO single dose or multiple dosing mode, change tendency with the dose and time characteristics of the factors of mouse movement research, the results showed that LO to normal mice slowed down when the length increases, has inhibitory effect on the motion; but it does not affect motor coordination in mice, showed improvement in promoting effect. Moreover, the effect of LO was significantly influenced by the dosage. LO did not change the normal motor function at low dose (0.35 g/kg), which proved the safety of LO as an intelligent drug. Finally, through the analysis of neurotransmitter and receptor expression, it is suggested that the change of exercise is related to the activity of dopaminergic nervous system and the level of 5-HT in the brain. In summary, this paper first from the nootropic effect and mechanism of action, "neurotoxic" aspects of lavender for a more comprehensive system research, the results show that LO has a puzzle effect obviously, and linalool in essential oil is the main active component; and the mechanism of antioxidation, anti apoptosis activity of intelligence, and the regulation of PI3K/Akt-Nrf2-HO-1 pathway; in addition, the LO variation caused by the movement of normal function in mice was significantly higher than the dose dose that puzzle.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
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本文编号：1343800