本文關(guān)鍵詞：薰衣草益智藥效組分及基于抗氧化作用的機(jī)制研究　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 薰衣草 精油 芳樟醇 阿爾茲海默病 學(xué)習(xí)記憶 抗氧化 神經(jīng)毒性

【摘要】：阿爾茲海默癥(Alzheimer's Disease,AD)是最常見的一種神經(jīng)退行性疾病,可導(dǎo)致認(rèn)知障礙、人格改變、行為異常等癥狀。氧化應(yīng)激與AD病理進(jìn)程密切相關(guān),AD中Aβ的沉積與氧化應(yīng)激相互誘導(dǎo),導(dǎo)致線粒體功能損傷,誘發(fā)神經(jīng)細(xì)胞凋亡。同時(shí)核因子 E2 相關(guān)因子 2(Nuclear factor-erythroid 2-related factor 2,Nrf2)-血紅素加氧酶-1(hemeoxygenase-1,HO-1)通路在機(jī)體抗氧化中發(fā)揮重要作用。所以,近年來氧化應(yīng)激所致?lián)p傷已置于AD研究的重要位置,并將從傳統(tǒng)天然藥物中尋找研發(fā)抗氧化活性物質(zhì)作為開發(fā)AD防治藥物的新途徑。薰衣草(Lavandula angustifolia Mill.)作為一種傳統(tǒng)中藥,能有效治療焦慮、抑郁、失眠、疼痛、驚厥、腦血栓等神經(jīng)系統(tǒng)疾病。薰衣草精油(Lavender oil,LO)及薰衣草水提物(Lavender extract,LE)都報(bào)道有中樞神經(jīng)系統(tǒng)活性,并表現(xiàn)出明顯的抗氧化作用。研究表明,主要成分(-)-芳樟醇(Linalool,LI)能通過調(diào)控Nrf2-HO-1通路發(fā)揮抗氧化、抗炎等作用。因此,我們推測(cè)薰衣草對(duì)AD學(xué)習(xí)記憶損傷有改善潛力。本論文擬通過多種體內(nèi)外模型對(duì)薰衣草的不同益智成分進(jìn)行學(xué)習(xí)記憶改善作用評(píng)價(jià),并從抗氧化入手探索相關(guān)的作用機(jī)制;此外,為了保證薰衣草用藥時(shí)的神經(jīng)系統(tǒng)安全性,本課題還擬研究薰衣草精油對(duì)正常動(dòng)物神經(jīng)行為,尤其是運(yùn)動(dòng)行為的影響,并對(duì)其相關(guān)作用機(jī)制進(jìn)行探索,為將薰衣草開發(fā)為AD防治藥物提供有效性、安全性的實(shí)驗(yàn)基礎(chǔ)和依據(jù)。主要研究?jī)?nèi)容如下:1.薰衣草益智活性成分的篩選及認(rèn)知改善作用評(píng)價(jià)首先對(duì)LO與LE神經(jīng)保護(hù)及益智作用進(jìn)行初步的評(píng)價(jià)及比較。通過H202、Aβ25-35誘導(dǎo)的PC12細(xì)胞損傷模型和東莨菪堿誘導(dǎo)認(rèn)知損傷小鼠模型,對(duì)LO和LE的神經(jīng)活性進(jìn)行初步評(píng)價(jià)。結(jié)果表明LO有明顯的神經(jīng)細(xì)胞損傷保護(hù)作用,并改善東莨菪堿導(dǎo)致的認(rèn)知障礙;而LE的體內(nèi)外藥效作用均不明顯,所以選擇LO做進(jìn)一步藥效研究。先建立GC-MS方法對(duì)薰衣草精油進(jìn)行成分確定和含量分析,發(fā)現(xiàn)其主要成分為芳樟醇(Linalool,LI)約為37.96%。接著同時(shí)對(duì)LO及LI的益智作用進(jìn)行研究,建立Aβ1-40海馬注射和D-galactose(D-gal)聯(lián)合AlC13誘導(dǎo)AD小鼠模型,水迷宮及避暗結(jié)果顯示,LO與LI都表現(xiàn)出明顯改善AD模型中學(xué)習(xí)記憶損傷的作用。2.薰衣草精油及芳樟醇基于抗氧化的神經(jīng)保護(hù)機(jī)制研究對(duì)于體內(nèi)模型,取Aβ1-40所致AD模型的海馬組織進(jìn)行生物化學(xué)、Western blot、染色分析發(fā)現(xiàn),LO或LI可以逆轉(zhuǎn)模型中抗氧化相關(guān)酶SOD和GPX活性的降低,和MDA的升高;并抑制caspase-9、caspase-3的激活,恢復(fù)MMP水平,使凋亡率明顯下降。同時(shí),在D-gal聯(lián)合AlCl3誘導(dǎo)模型海馬中,LO或LI同樣表現(xiàn)了抗氧化的作用,并且提高神經(jīng)可塑性相關(guān)蛋白CaMKII,p-CaMKII,BDNF和TrkB的表達(dá)。此外,對(duì)于H2O2、Aβ25-35誘導(dǎo)的體外模型,LI可以逆轉(zhuǎn)ROS、NO的水平、caspase-9、caspase-3活性、MMP和細(xì)胞凋亡率。所以,LO與LI在神經(jīng)保護(hù)中表現(xiàn)出明顯的抗氧化、抗凋亡的作用。進(jìn)一步對(duì)Nrf2-HO-1通路在LI抗氧化神經(jīng)保護(hù)中的作用進(jìn)行研究。將LI對(duì)PC12細(xì)胞進(jìn)行直接干預(yù),Western blot結(jié)果表明:LI可以時(shí)間依賴和濃度依賴地促進(jìn)HO-1、Nrf2的表達(dá)及Akt的激活,并且促進(jìn)Nrf2由胞質(zhì)到胞內(nèi)的轉(zhuǎn)移;當(dāng)使用PI3K/Akt抑制劑LY249002或HO-1的抑制劑ZnPP對(duì)細(xì)胞進(jìn)行預(yù)處理后,LI所誘導(dǎo)的HO高表達(dá)受到抑制,并且LI對(duì)抗氧化的保護(hù)作用也被破壞。此外,LI也使Aβ1-40和D-gal+AlCl3誘導(dǎo)小鼠海馬中HO-1、Nrf2的表達(dá)升高�？梢酝茢�,LI對(duì)神經(jīng)細(xì)胞抗氧化保護(hù)作用可能是通過PI3K/Akt-Nrf2-HO-1通路介導(dǎo)。3.薰衣草精油對(duì)正常小鼠神經(jīng)行為的影響先通過一系列行為學(xué)實(shí)驗(yàn)觀察到LO對(duì)正常大鼠的運(yùn)動(dòng)能力有明顯的抑制作用,對(duì)情緒和學(xué)習(xí)記憶能力影響不顯著。進(jìn)而設(shè)置LO單次給藥或多次給藥模式,研究小鼠運(yùn)動(dòng)特征隨給藥劑量和時(shí)間因素的變化趨勢(shì),結(jié)果表明LO使正常小鼠運(yùn)動(dòng)速度減慢,時(shí)長(zhǎng)增加,對(duì)其運(yùn)動(dòng)有抑制作用;但并不影響小鼠的運(yùn)動(dòng)協(xié)調(diào)性,表現(xiàn)出改善促進(jìn)作用。并且LO的作用受給藥劑量的影響較為顯著,在低劑量(0.35 g/kg)時(shí)LO不改變正常的運(yùn)動(dòng)功能,證明了 LO作為益智藥物應(yīng)用的安全性。最后通過神經(jīng)遞質(zhì)、受體表達(dá)分析,推測(cè)運(yùn)動(dòng)的變化與腦內(nèi)多巴胺能神經(jīng)系統(tǒng)活性及5-HT水平相關(guān)。綜上所述,本課題首次從益智作用、作用機(jī)制、“神經(jīng)毒性”等多個(gè)方面對(duì)薰衣草進(jìn)行了較全面系統(tǒng)的研究,結(jié)果表明LO有明顯的益智作用,而芳樟醇為精油中的主要活性成分;這種益智作用的機(jī)制與抗氧化、抗凋亡的活性相關(guān),并受PI3K/Akt-Nrf2-HO-1通路的調(diào)控;此外,LO引起正常小鼠運(yùn)動(dòng)功能變化的劑量顯著地高于益智藥效劑量,說明了 LO作為益智藥物使用的安全性。本課題的研究將為從薰衣草中開發(fā)AD潛在預(yù)防治療藥物提供更多的實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5
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本文編號(hào)：1343800