本文關(guān)鍵詞：別構(gòu)調(diào)節(jié)sigma-1受體的抗抑郁作用及其機(jī)制研究　出處：《蘇州大學(xué)》2016年博士論文　論文類型：學(xué)位論文

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【摘要】：第一部分SOMCL-668具有別構(gòu)調(diào)節(jié)sigma-1受體的活性目的:Sigma-1受體在抑郁癥等眾多神經(jīng)精神疾病治療中有重要作用,同時(shí),別構(gòu)調(diào)節(jié)是調(diào)節(jié)受體功能重要途徑之一。因此,別構(gòu)調(diào)節(jié)sigma-1可能成為抑郁癥治療的新靶點(diǎn)。前期競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)表明,新型化合物3-methyl-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ol(SOMCL-668)能以別構(gòu)調(diào)節(jié)的方式促進(jìn)sigma-1受體特異性激動(dòng)劑3H-(+)-pentazocine與sigma-1受體的結(jié)合。在此研究基礎(chǔ)上,本部分將從生物學(xué)功能角度進(jìn)一步證實(shí)SOMCL-668的別構(gòu)調(diào)節(jié)功能,從而為研究別構(gòu)調(diào)節(jié)sigma-1受體的作用提供有力支持。方法:培養(yǎng)HT22小鼠海馬神經(jīng)元細(xì)胞系,采用細(xì)胞免疫熒光法,檢測(cè)SOMCL-668單獨(dú)或聯(lián)用sigma-1受體激動(dòng)劑(+)SKF-10047對(duì)sigma-1受體與Bip(binding immunoglobulin protein)蛋白共定位的影響;采用免疫共沉淀方法,檢測(cè)SOMCL-668單獨(dú)或聯(lián)用sigma-1受體激動(dòng)劑(+)SKF-10047對(duì)sigma-1受體與Bip結(jié)合情況的影響;分離細(xì)胞膜成分,檢測(cè)SOMCL-668單獨(dú)或聯(lián)用sigma-1受體激動(dòng)劑(+)SKF-10047對(duì)sigma-1受體轉(zhuǎn)位現(xiàn)象的影響;培養(yǎng)原代神經(jīng)元,檢測(cè)SOMCL-668單獨(dú)或聯(lián)用sigma-1受體激動(dòng)劑(+)SKF-10047對(duì)神經(jīng)元神經(jīng)突起生長(zhǎng)以及BDNF(brain-derived neurotrophic factor,腦源性神經(jīng)營(yíng)養(yǎng)因子)分泌的影響。結(jié)果:SOMCL-668本身不能改變sigma-1受體和Bip的共定位或結(jié)合,但是可以明顯促進(jìn)激動(dòng)劑誘導(dǎo)的sigma-1受體與Bip的分離;同時(shí),SOMCL-668單獨(dú)不能,但聯(lián)用激動(dòng)劑后顯著增加sigma-1受體向細(xì)胞膜的轉(zhuǎn)位;此外,SOMCL-668自身不具備,但是可以顯著增加激動(dòng)劑誘導(dǎo)的神經(jīng)突起生長(zhǎng)和BDNF分泌的能力。結(jié)論:SOMCL-668具備別構(gòu)調(diào)節(jié)sigma-1受體的活性,是一種新型的sigma-1受體別構(gòu)調(diào)節(jié)劑。第二部分別構(gòu)調(diào)節(jié)sigma-1受體具有抗抑郁作用目的:本部分將以SOMCL-668為工具,重點(diǎn)考察別構(gòu)調(diào)節(jié)sigma-1受體是否產(chǎn)生抗抑郁作用,并探討其可能的抗抑郁作用機(jī)制。方法:(1)采用小鼠強(qiáng)迫游泳、懸尾實(shí)驗(yàn)等急性抗抑郁藥物篩選模型初步探討SOMCL-668的抗抑郁作用;(2)采用慢性不可預(yù)知性溫和刺激制作抑郁癥模型,糖水偏好、曠場(chǎng)實(shí)驗(yàn)等檢測(cè)SOMCL-668的抗抑郁作用;免疫印跡法檢測(cè)SOMCL-668對(duì)抑郁癥中BDNF-GSK3β(glycogen synthase kinase 3β,糖原合成激酶3β)通路的影響變化;(3)采用免疫印跡法,同時(shí)應(yīng)用藥理學(xué)及分子生物學(xué)手段檢測(cè)sigma-1受體對(duì)GSK3β活性的抑制作用及其調(diào)控機(jī)制;(4)采用免疫印跡法,從動(dòng)物及細(xì)胞水平觀察SOMCL-668對(duì)GSK3β活性的抑制作用;采用藥理學(xué)手段間接激活GSK3β,考察GSK3β在SOMCL-668的抗抑郁效應(yīng)中的作用。結(jié)果:(1)SOMCL-668可以減少?gòu)?qiáng)迫游泳、懸尾實(shí)驗(yàn)中小鼠不動(dòng)時(shí)間,表現(xiàn)出抗抑郁作用,提前給以sigma-1受體拮抗劑BD1047則取消其抗抑郁作用;(2)SOMCL-668可以改善抑郁癥模型中小鼠快感缺乏、焦慮及體重下降等癥狀,同時(shí)可以緩解抑郁癥導(dǎo)致的BDNF及GSK3β(ser-9)磷酸化水平的下調(diào),并且SOMCL-668抗抑郁起效時(shí)間(1周內(nèi))優(yōu)于現(xiàn)有抗抑郁藥物文拉法辛。(3)給予sigma-1受體激動(dòng)劑或過(guò)表達(dá)sigma-1受體均能增加GSK3β(ser-9)磷酸化水平(即抑制其活性),且Trk B受體抑制劑、AKT通路抑制劑可以拮抗sigma-1受體對(duì)GSK3β(ser-9)磷酸化的影響。結(jié)果證實(shí)sigma-1受體可以調(diào)控GSK3β活性。(4)在動(dòng)物實(shí)驗(yàn)中,SOMCL-668可以快速增加小鼠海馬中GSK3β(ser-9)磷酸化水平,sigma-1受體拮抗劑BD1047則阻斷這種作用;在細(xì)胞實(shí)驗(yàn)中,SOMCL-668可以通過(guò)別構(gòu)調(diào)節(jié)的方式增加HT22細(xì)胞中GSK3β(ser-9)磷酸化水平,siRNA沉默sigma-1受體表達(dá)后同樣取消這種作用。藥理學(xué)激活GSK3β可以阻斷SOMCL-668在強(qiáng)迫游泳及懸尾實(shí)驗(yàn)中的抗抑郁作用。結(jié)論:sigma-1受體別構(gòu)調(diào)節(jié)劑SOMCL-668具有快速的抗抑郁作用,其抗抑郁作用可能和BDNF-GSK3β通路有關(guān),這將為抗抑郁藥物研發(fā)提供新的策略。
[Abstract]:The first part is the SOMCL-668 allosteric activity to modulate sigma-1 receptors: Sigma-1 receptors play an important role in the treatment of depression and other mental illness at the same time, allosteric regulation is one of the important approaches to regulate receptor function. Therefore, the allosteric regulation of sigma-1 may become a new target for the treatment of depression. The competitive binding experiments show that the new compound 3-methyl-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ol (SOMCL-668) can promote sigma-1 receptor specific agonist 3H- in allosteric manner (+) -pentazocine and sigma-1 receptor binding. On the basis of this research, this part from the perspective of the biological function of SOMCL-668 confirm the allosteric regulation function, which provides strong support for the study of the allosteric regulation of sigma-1 receptor the effect. Methods: HT22 mice and cultured hippocampal neurons cell lines by cell immunofluorescence method to detect SOM CL-668 alone or in combination with sigma-1 receptor agonist SKF-10047 on sigma-1 receptor (+) and Bip (binding immunoglobulin protein) protein co localization; CO immunoprecipitation method, detection of SOMCL-668 alone or in combination with sigma-1 receptor agonist (+) SKF-10047 was affected by the combination of Bip and sigma-1; separation of cell membrane components the detection of SOMCL-668, either alone or in combination with sigma-1 (+) SKF-10047 receptor agonist effect on sigma-1 receptor translocation; primary cultured neurons, the detection of SOMCL-668 alone or in combination with sigma-1 (+) receptor agonist SKF-10047 on neuronal neurite growth and BDNF (brain-derived neurotrophic factor, brain-derived neurotrophic factor) secretion results: SOMCL-668 itself cannot change the sigma-1 receptor and Bip co localization or combination, but can significantly promote the separation of agonist induced receptor sigma-1 and Bip; and When SOMCL-668 alone cannot, but combined with sigma-1 receptor agonists increased significantly after translocation to the cell membrane; in addition, the SOMCL-668 itself does not have, but can significantly increase the ability of agonist induced neurite growth and secretion of BDNF. Conclusion: SOMCL-668 has the allosteric regulation of sigma-1 receptor activity, is a new type of sigma-1 receptor allosteric modulators. The second part of the allosteric regulation of sigma-1 receptor with antidepressant effect Objective: this part by means of SOMCL-668, focuses on the allosteric regulation of sigma-1 receptors could produce antidepressant effect, and investigate the possible mechanism of antidepressant effect. Methods: (1) by the forced swimming test, tail suspension test and acute antidepressant drug screening model to explore the antidepressant effect of SOMCL-668; (2) the chronic unpredictable mild stress depression model making, sucrose preference, open field test SOMCL-66 8 of the antidepressant effect; Western blot detection of SOMCL-668 on depression (glycogen synthase kinase BDNF-GSK3 beta 3 beta, glycogen synthase kinase 3 beta) signaling pathway changes; (3) by Western blot, and the application of pharmacology and molecular biology method to detect sigma-1 receptor inhibition of GSK3 beta activity and its regulation mechanism; (4) by Western blot, the inhibition effect of SOMCL-668 on the cellular level and animal GSK3 beta activity; indirect activation of GSK3 beta pharmacology by means of investigation, the role of GSK3 beta on the antidepressant effect of SOMCL-668. Results: (1) SOMCL-668 can reduce the forced swimming, tail suspension test in mice immobility time. Show the antidepressant effect of sigma-1 receptor antagonist BD1047 in advance to cancel its antidepressant effect; (2) SOMCL-668 can improve the mice model of depression in the lack of pleasure, anxiety and weight loss and other symptoms, at the same time Can ease depression in BDNF and GSK3 beta (ser-9) lowered the level of phosphate, SOMCL-668 and anti depression onset time (1 weeks) than existing antidepressant venlafaxine. (3) administration of sigma-1 receptor agonist or overexpression of sigma-1 receptor can increase GSK3 beta (ser-9) phosphorylation level (i.e. inhibition the activity of Trk and B), receptor inhibitor, AKT pathway inhibitors can antagonize the sigma-1 receptor of GSK3 beta (ser-9) phosphorylation. The results indicate that the sigma-1 receptor regulates GSK3 activity. (4) in animal experiments, SOMCL-668 can quickly increase mouse hippocampus GSK3 beta (ser-9) phosphorylation of sigma-1 receptor. BD1047 antagonist blocked this effect; in vitro experiments, SOMCL-668 through allosteric way to increase HT22 cell GSK3 beta (ser-9) phosphorylation, sigma-1 receptor expression after siRNA silencing also canceled this effect. Pharmacology The activation of GSK3 beta SOMCL-668 can be blocked by the forced swimming test and the tail suspension test in the antidepressant effect. Conclusion: the sigma-1 receptor allosteric regulator SOMCL-668 has rapid antidepressant effect, its antidepressant and BDNF-GSK3 beta signaling pathway, which will provide a new strategy for the development of antidepressant drugs.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96

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