别构调节sigma-1受体的抗抑郁作用及其机制研究

发布时间：2018-01-14 15:45

本文关键词：别构调节sigma-1受体的抗抑郁作用及其机制研究　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分SOMCL-668具有别构调节sigma-1受体的活性目的:Sigma-1受体在抑郁症等众多神经精神疾病治疗中有重要作用,同时,别构调节是调节受体功能重要途径之一。因此,别构调节sigma-1可能成为抑郁症治疗的新靶点。前期竞争结合实验表明,新型化合物3-methyl-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ol(SOMCL-668)能以别构调节的方式促进sigma-1受体特异性激动剂3H-(+)-pentazocine与sigma-1受体的结合。在此研究基础上,本部分将从生物学功能角度进一步证实SOMCL-668的别构调节功能,从而为研究别构调节sigma-1受体的作用提供有力支持。方法:培养HT22小鼠海马神经元细胞系,采用细胞免疫荧光法,检测SOMCL-668单独或联用sigma-1受体激动剂(+)SKF-10047对sigma-1受体与Bip(binding immunoglobulin protein)蛋白共定位的影响;采用免疫共沉淀方法,检测SOMCL-668单独或联用sigma-1受体激动剂(+)SKF-10047对sigma-1受体与Bip结合情况的影响;分离细胞膜成分,检测SOMCL-668单独或联用sigma-1受体激动剂(+)SKF-10047对sigma-1受体转位现象的影响;培养原代神经元,检测SOMCL-668单独或联用sigma-1受体激动剂(+)SKF-10047对神经元神经突起生长以及BDNF(brain-derived neurotrophic factor,脑源性神经营养因子)分泌的影响。结果:SOMCL-668本身不能改变sigma-1受体和Bip的共定位或结合,但是可以明显促进激动剂诱导的sigma-1受体与Bip的分离;同时,SOMCL-668单独不能,但联用激动剂后显著增加sigma-1受体向细胞膜的转位;此外,SOMCL-668自身不具备,但是可以显著增加激动剂诱导的神经突起生长和BDNF分泌的能力。结论:SOMCL-668具备别构调节sigma-1受体的活性,是一种新型的sigma-1受体别构调节剂。第二部分别构调节sigma-1受体具有抗抑郁作用目的:本部分将以SOMCL-668为工具,重点考察别构调节sigma-1受体是否产生抗抑郁作用,并探讨其可能的抗抑郁作用机制。方法:(1)采用小鼠强迫游泳、悬尾实验等急性抗抑郁药物筛选模型初步探讨SOMCL-668的抗抑郁作用;(2)采用慢性不可预知性温和刺激制作抑郁症模型,糖水偏好、旷场实验等检测SOMCL-668的抗抑郁作用;免疫印迹法检测SOMCL-668对抑郁症中BDNF-GSK3β(glycogen synthase kinase 3β,糖原合成激酶3β)通路的影响变化;(3)采用免疫印迹法,同时应用药理学及分子生物学手段检测sigma-1受体对GSK3β活性的抑制作用及其调控机制;(4)采用免疫印迹法,从动物及细胞水平观察SOMCL-668对GSK3β活性的抑制作用;采用药理学手段间接激活GSK3β,考察GSK3β在SOMCL-668的抗抑郁效应中的作用。结果:(1)SOMCL-668可以减少强迫游泳、悬尾实验中小鼠不动时间,表现出抗抑郁作用,提前给以sigma-1受体拮抗剂BD1047则取消其抗抑郁作用;(2)SOMCL-668可以改善抑郁症模型中小鼠快感缺乏、焦虑及体重下降等症状,同时可以缓解抑郁症导致的BDNF及GSK3β(ser-9)磷酸化水平的下调,并且SOMCL-668抗抑郁起效时间(1周内)优于现有抗抑郁药物文拉法辛。(3)给予sigma-1受体激动剂或过表达sigma-1受体均能增加GSK3β(ser-9)磷酸化水平(即抑制其活性),且Trk B受体抑制剂、AKT通路抑制剂可以拮抗sigma-1受体对GSK3β(ser-9)磷酸化的影响。结果证实sigma-1受体可以调控GSK3β活性。(4)在动物实验中,SOMCL-668可以快速增加小鼠海马中GSK3β(ser-9)磷酸化水平,sigma-1受体拮抗剂BD1047则阻断这种作用;在细胞实验中,SOMCL-668可以通过别构调节的方式增加HT22细胞中GSK3β(ser-9)磷酸化水平,siRNA沉默sigma-1受体表达后同样取消这种作用。药理学激活GSK3β可以阻断SOMCL-668在强迫游泳及悬尾实验中的抗抑郁作用。结论:sigma-1受体别构调节剂SOMCL-668具有快速的抗抑郁作用,其抗抑郁作用可能和BDNF-GSK3β通路有关,这将为抗抑郁药物研发提供新的策略。
[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.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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