GSK3β过表达对阿姆西

发布时间：2018-06-26 02:58

本文选题：阿姆西汀 + 小补心汤总黄酮　；参考：《安徽理工大学》2017年硕士论文

【摘要】：目的:军事医学科学院毒物药物研究所神经精神药理研究室长期从事抗抑郁药物研发及相关基础研究。前期以度洛西汀(duloxetine,DLX)为先导化合物,合成筛选出新型化合物盐酸阿姆西汀(ammoxetine,AMX),前期已经证实其具有显著的抗抑郁活性,并且肝毒性远远低于度洛西汀。小补心汤(XBXT)属于传统古方中药,最早见于南北朝时期的敦煌莫高窟遗书《辅行决脏腑用药法要》,由代赭石、旋复花、竹叶、淡豆豉四味中药组成。该研究室首次发现XBXT及其总黄酮提取物(XBXT-2)在多种模型上表现出明确的抗抑郁活性。近年来,研究发现糖原合成酶激酶3β(glycogen synthase kinase,GSK3β)及其相关信号通路与抑郁症的发生和治疗密切相关,GSK3β失活(磷酸化增加)会产生抗抑郁作用,本课题组前期实验发现AMX和XBXT-2均可以增加GSK3β磷酸化水平并激活其相关信号通路。本研究拟在前期工作的基础上,采用基因转染技术,使小鼠脑组织海马部位GSK3β过表达或者持续激活,进一步探究AMX和XBXT-2对GSK3β相关信号通路的影响,试图阐明GSK3β及其相关信号通路是否是AMX和XBXT-2抗抑郁作用发挥的关键环节。方法:本研究采用以腺相关病毒(adeno-associatedvirus,AAV)为载体的基因转染技术,构建包装有GSK3β(WT)野生型和GSK3β(S9A)突变型基因的腺相关病毒,通过海马立体定位微注射的方式,使GSK3β在小鼠海马过表达或者持续激活,通过一系列行为学和分子生物学实验探讨GSK3β是否是AMX、XBXT-2发挥抗抑郁、抗焦虑作用的关键分子靶标。(一)海马GSK3β过表达/持续激活对AMX抗抑郁作用的影响:进行小鼠海马脑区微注射,使GSK3β在海马过表达/持续激活,通过悬尾实验和强迫游泳实验,研究海马GSK3β过表达/持续激活对阿姆西汀抗抑郁作用的影响;并通过蛋白免疫印迹法(western blot)检测海马脑区PI-3K/Akt/GSK3β信号通路的功能蛋白及脑源性神经营养因子(BDNF)含量的变化。(二)海马GSK3β持续激活对XBXT-2抗抑郁作用的影响:进行小鼠海马脑区微注射,使GSK3β在海马持续激活,通过悬尾实验、强迫游泳实验、高架十字迷宫实验、爬梯实验研究海马GSK3β持续激活对小补心汤总黄酮抗抑郁、抗焦虑作用的影响;进行小鼠海马脑区微注射,使GSK3β在海马持续激活,在获得性无助模型上探究GSK3β的失抑制是否会增加抑郁的易感性;并通过western bolt方法检测PI-3K/Akt/GSK3β信号通路各蛋白以及BDNF含量的变化,探讨GSK3β失抑制增加抑郁易感性的机制。结果:(一)AMX单次给药(2.5mg/kg)可以显著缩短阴性病毒组小鼠的悬尾不动时间,该行为效应能够被小鼠海马GSK3β过表达/持续激活所取消;AMX单次给药(5mg/kg)可以显著缩短阴性病毒组小鼠的强迫游泳不动时间,该行为效应能够被小鼠海马GSK3β过表达/持续激活所取消;无论是药物还是海马GSK3β过表达/持续激活均对小鼠自发活动没有影响;western blot结果显示AMX可以上调p-Akt、p-GSK3β、p-CREB、BDNF的蛋白表达水平,但是该作用被GSK3β过表达/持续激活所取消。(二)XBXT-2长期给药(100mg/kg)可以显著缩短阴性病毒组小鼠的悬尾不动时间和强迫游泳不动时间,该行为效应均能够被海马GSK3β持续激活所取消;XBXT-2长期给药(100mg/kg),可以显著缩短阴性病毒组小鼠在爬梯实验中的站立次数,该行为效应能够被海马GSK3β持续激活取消;XBXT-2长期给药(100mg/kg),可以显著增加阴性病毒组小鼠在高架实验中进入开臂次数的百分比和在开臂停留时间的百分比,该行为效应不仅被海马GSK3β持续激活所取消,并且海马GSK3β持续激活显著降低了小鼠进入开臂次数的百分比;在获得性无助模型中,GSK3β持续激活小鼠逃避失败次数显著增加,糖水偏嗜度显著下降,并且XBXT-2的抗抑郁行为效应被完全取消;western blot结果显示在获得性无助造模之后,GSK3β持续激活小鼠的p-Akt、p-GSK3β、p-CREB、BDNF表达水平同阴性病毒组相比均显著下降,并且XBXT-2对这些蛋白的上调作用亦被完全取消。结论:通过本课题实验研究可得到如下结论:(1)在悬尾实验和强迫游泳实验中,海马GSK3β过表达或持续激活不能引起小鼠抑郁样表现,但是能够取消AMX和XBXT-2的抗抑郁行为效应;(2)在高架十字迷宫实验和爬梯实验上,GSK3β持续激活取消了 XBXT-2的抗焦虑作用,并且在高架十字迷宫实验中产生致焦虑样行为表现;(3)在获得性无助模型中,GSK3β持续激活会导致应激易感性增加;(4)GSK3β过表达/持续激活对p-Akt、p-CREB和BDNF的蛋白表达水平无显著影响,但是能够取消AMX对这些蛋白含量的上调作用;(5)GSK3β持续激活在获得性无助模型之后,能够显著下调p-Akt、p-CREB和BDNF的蛋白表达水平,并取消了 XBXT-2对这些蛋白水平的上调作用。
[Abstract]:Objective: the neuropsychiatry Department of the Institute of toxicology and drug research, Military Medical Science Academy of the PLA, has been engaged in the research and development of antidepressant drugs for a long time and related basic research. In the early stage, duloxetine (DLX) was used as the precursor compound, and the new compound was synthesized and screened for ammoxetine (AMX), and it has been proved to have significant antidepressant in the early stage. Activity, and hepatotoxicity is far lower than duloxetine. Xiao Bu Xin Tang (XBXT) belongs to traditional traditional Chinese medicine. It was first seen in the Dunhuang Mogao Grottoes of the northern and Southern Dynasties, which was composed of four traditional Chinese herbs, ochre, flower, bamboo leaves and fermented soya bean. The XBXT and its total flavonoids extract (XBXT-2) were first found in the laboratory. In recent years, it has been found that glycogen synthetase kinase 3 beta (glycogen synthase kinase, GSK3 beta) and its related signaling pathways are closely related to the occurrence and treatment of depression. GSK3 beta inactivation (phosphorylation increases) will produce antidepressant use. Earlier experiments in our group found that AMX and XBXT-2 could be increased. On the basis of earlier work, this study intends to use gene transfection on the basis of early work to make GSK3 beta over expression or continuous activation in the hippocampus of the brain tissue of mice, to further explore the effect of AMX and XBXT-2 on the GSK3 beta related signaling pathway, and to try to clarify whether GSK3 beta and its related signaling pathways are or not. The key link of the antidepressant effect of AMX and XBXT-2. Methods: in this study, the gene transfection technology using adeno-associatedvirus (AAV) as the carrier was used to construct adeno-related virus packed with GSK3 beta (WT) wild type and GSK3 beta (S9A) mutant gene, and GSK3 beta in the hippocampus of mice by stereotaxic microinjection of hippocampus. Expression or continuous activation, through a series of behavioural and molecular biological experiments to explore whether GSK3 beta is AMX, XBXT-2 plays the key molecular target of antidepressant and anti anxiety effects. (1) the effect of GSK3 beta overexpression / continuous activation on the antidepressant effect of AMX: microinjection of the hippocampus in the hippocampus of mice to make GSK3 beta in the hippocampus overexpressed / sustained excitation The effect of overexpression / continuous activation of GSK3 beta on the antidepressant effect of amsietine was studied by hanging tail experiment and forced swimming test. The changes in the functional protein of PI-3K/Akt/GSK3 beta signaling pathway in the hippocampus and the content of BDNF in the hippocampus were detected by Western blot. (two) hippocampus GSK3 beta The effect of continuous activation on the antidepressant effect of XBXT-2: microinjection of hippocampal brain in mice to enable GSK3 beta to continue to activate in the hippocampus, through tail suspension experiment, forced swimming test, elevated cross maze test, and climbing ladder experiment to study the effect of GSK3 beta continuous activation of hippocampal GSK3 on the depression and anxiety of Xiao Bu Xin Tang, and to carry out the hippocampus brain area of mice. Microinjection makes GSK3 beta continuously activated in the hippocampus. In the acquired helplessness model, it is found that the depressive susceptibility of GSK3 beta may increase the susceptibility to depression; and the changes in the protein and BDNF content of the PI-3K/Akt/GSK3 beta signaling pathway are detected by the Western bolt method. The mechanism of GSK3 beta inhibition to increase the susceptibility to depression is explored. Results: (1) single dose of AMX The drug (2.5mg/kg) can significantly shorten the time of tail suspension in the negative virus group, which can be cancelled by the overexpression / continuous activation of GSK3 beta in the hippocampus of mice. The single dose of AMX (5mg/kg) can significantly shorten the time of forced swimming in the negative virus group, and the behavioral effect can be overexpressed and sustained by the GSK3 beta in the hippocampus of the mice. GSK3 beta overexpression / Western blot results showed that AMX could up regulate the protein expression level of p-Akt, p-GSK3 beta, p-CREB, BDNF, but the effect was cancelled by GSK3 beta overexpression / continuous activation. (two) XBXT-2 long term delivery (100mg/kg) could be significantly shortened. The action effect can be cancelled by the continuous activation of hippocampal GSK3 beta, and XBXT-2 long-term administration (100mg/kg) can significantly shorten the standing times of the negative virus group in the climbing ladder experiment, and the behavioral effect can be cancelled by the GSK3 beta in the hippocampus; XBXT-2 is long. 100mg/kg could significantly increase the percentage of the number of open arms in the viaduct mice and the percentage of the opening time in the open arm. The behavioral effect was not only cancelled by the sustained activation of GSK3 beta in the hippocampus, but the continued activation of GSK3 beta in the hippocampus significantly reduced the percentage of the mice entering the number of open arms. In the model, the number of GSK3 beta continuous activation in mice increased significantly, the sugar water bias decreased significantly, and the antidepressant effect of XBXT-2 was completely cancelled. The Western blot results showed that after the acquired helplessness model, GSK3 beta continued to activate the p-Akt in mice, p-GSK3 beta, p-CREB, and BDNF expression levels were compared with those of the negative virus group. The up-regulation effect of XBXT-2 on these proteins was completely cancelled. Conclusion: the following conclusions are obtained: (1) in the tail suspension experiment and forced swimming test, the overexpression or continuous activation of GSK3 beta in the hippocampus can not cause depressive behavior in mice, but it can abolish the antidepressant effect of AMX and XBXT-2; (2) The continuous activation of GSK3 beta cancels the anti anxiety effect of XBXT-2 and produces anxiety like behavior in the elevated cross labyrinth experiment. (3) in the acquired helplessness model, the continuous activation of GSK3 beta leads to the increase of stress susceptibility; (4) GSK3 beta overexpression / continuous activation of p-Akt, p-CREB and BDNF There was no significant effect of protein expression level, but it could eliminate the up-regulation effect of AMX on these proteins. (5) after the GSK3 beta was activated in the acquired helplessness model, it could significantly reduce the protein expression level of p-Akt, p-CREB and BDNF, and abolished the up-regulation effect of XBXT-2 on these protein levels.
【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285;R96

【参考文献】