本文選題：阿姆西汀 + 小補心湯總黃酮��； 參考：《安徽理工大學》2017年碩士論文

【摘要】：目的:軍事醫(yī)學科學院毒物藥物研究所神經(jīng)精神藥理研究室長期從事抗抑郁藥物研發(fā)及相關基礎研究。前期以度洛西汀(duloxetine,DLX)為先導化合物,合成篩選出新型化合物鹽酸阿姆西汀(ammoxetine,AMX),前期已經(jīng)證實其具有顯著的抗抑郁活性,并且肝毒性遠遠低于度洛西汀。小補心湯(XBXT)屬于傳統(tǒng)古方中藥,最早見于南北朝時期的敦煌莫高窟遺書《輔行決臟腑用藥法要》,由代赭石、旋復花、竹葉、淡豆豉四味中藥組成。該研究室首次發(fā)現(xiàn)XBXT及其總黃酮提取物(XBXT-2)在多種模型上表現(xiàn)出明確的抗抑郁活性。近年來,研究發(fā)現(xiàn)糖原合成酶激酶3β(glycogen synthase kinase,GSK3β)及其相關信號通路與抑郁癥的發(fā)生和治療密切相關,GSK3β失活(磷酸化增加)會產(chǎn)生抗抑郁作用,本課題組前期實驗發(fā)現(xiàn)AMX和XBXT-2均可以增加GSK3β磷酸化水平并激活其相關信號通路。本研究擬在前期工作的基礎上,采用基因轉染技術,使小鼠腦組織海馬部位GSK3β過表達或者持續(xù)激活,進一步探究AMX和XBXT-2對GSK3β相關信號通路的影響,試圖闡明GSK3β及其相關信號通路是否是AMX和XBXT-2抗抑郁作用發(fā)揮的關鍵環(huán)節(jié)。方法:本研究采用以腺相關病毒(adeno-associatedvirus,AAV)為載體的基因轉染技術,構建包裝有GSK3β(WT)野生型和GSK3β(S9A)突變型基因的腺相關病毒,通過海馬立體定位微注射的方式,使GSK3β在小鼠海馬過表達或者持續(xù)激活,通過一系列行為學和分子生物學實驗探討GSK3β是否是AMX、XBXT-2發(fā)揮抗抑郁、抗焦慮作用的關鍵分子靶標。(一)海馬GSK3β過表達/持續(xù)激活對AMX抗抑郁作用的影響:進行小鼠海馬腦區(qū)微注射,使GSK3β在海馬過表達/持續(xù)激活,通過懸尾實驗和強迫游泳實驗,研究海馬GSK3β過表達/持續(xù)激活對阿姆西汀抗抑郁作用的影響;并通過蛋白免疫印跡法(western blot)檢測海馬腦區(qū)PI-3K/Akt/GSK3β信號通路的功能蛋白及腦源性神經(jīng)營養(yǎng)因子(BDNF)含量的變化。(二)海馬GSK3β持續(xù)激活對XBXT-2抗抑郁作用的影響:進行小鼠海馬腦區(qū)微注射,使GSK3β在海馬持續(xù)激活,通過懸尾實驗、強迫游泳實驗、高架十字迷宮實驗、爬梯實驗研究海馬GSK3β持續(xù)激活對小補心湯總黃酮抗抑郁、抗焦慮作用的影響;進行小鼠海馬腦區(qū)微注射,使GSK3β在海馬持續(xù)激活,在獲得性無助模型上探究GSK3β的失抑制是否會增加抑郁的易感性;并通過western bolt方法檢測PI-3K/Akt/GSK3β信號通路各蛋白以及BDNF含量的變化,探討GSK3β失抑制增加抑郁易感性的機制。結果:(一)AMX單次給藥(2.5mg/kg)可以顯著縮短陰性病毒組小鼠的懸尾不動時間,該行為效應能夠被小鼠海馬GSK3β過表達/持續(xù)激活所取消;AMX單次給藥(5mg/kg)可以顯著縮短陰性病毒組小鼠的強迫游泳不動時間,該行為效應能夠被小鼠海馬GSK3β過表達/持續(xù)激活所取消;無論是藥物還是海馬GSK3β過表達/持續(xù)激活均對小鼠自發(fā)活動沒有影響;western blot結果顯示AMX可以上調(diào)p-Akt、p-GSK3β、p-CREB、BDNF的蛋白表達水平,但是該作用被GSK3β過表達/持續(xù)激活所取消。(二)XBXT-2長期給藥(100mg/kg)可以顯著縮短陰性病毒組小鼠的懸尾不動時間和強迫游泳不動時間,該行為效應均能夠被海馬GSK3β持續(xù)激活所取消;XBXT-2長期給藥(100mg/kg),可以顯著縮短陰性病毒組小鼠在爬梯實驗中的站立次數(shù),該行為效應能夠被海馬GSK3β持續(xù)激活取消;XBXT-2長期給藥(100mg/kg),可以顯著增加陰性病毒組小鼠在高架實驗中進入開臂次數(shù)的百分比和在開臂停留時間的百分比,該行為效應不僅被海馬GSK3β持續(xù)激活所取消,并且海馬GSK3β持續(xù)激活顯著降低了小鼠進入開臂次數(shù)的百分比;在獲得性無助模型中,GSK3β持續(xù)激活小鼠逃避失敗次數(shù)顯著增加,糖水偏嗜度顯著下降,并且XBXT-2的抗抑郁行為效應被完全取消;western blot結果顯示在獲得性無助造模之后,GSK3β持續(xù)激活小鼠的p-Akt、p-GSK3β、p-CREB、BDNF表達水平同陰性病毒組相比均顯著下降,并且XBXT-2對這些蛋白的上調(diào)作用亦被完全取消。結論:通過本課題實驗研究可得到如下結論:(1)在懸尾實驗和強迫游泳實驗中,海馬GSK3β過表達或持續(xù)激活不能引起小鼠抑郁樣表現(xiàn),但是能夠取消AMX和XBXT-2的抗抑郁行為效應;(2)在高架十字迷宮實驗和爬梯實驗上,GSK3β持續(xù)激活取消了 XBXT-2的抗焦慮作用,并且在高架十字迷宮實驗中產(chǎn)生致焦慮樣行為表現(xiàn);(3)在獲得性無助模型中,GSK3β持續(xù)激活會導致應激易感性增加;(4)GSK3β過表達/持續(xù)激活對p-Akt、p-CREB和BDNF的蛋白表達水平無顯著影響,但是能夠取消AMX對這些蛋白含量的上調(diào)作用;(5)GSK3β持續(xù)激活在獲得性無助模型之后,能夠顯著下調(diào)p-Akt、p-CREB和BDNF的蛋白表達水平,并取消了 XBXT-2對這些蛋白水平的上調(diào)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.
【學位授予單位】：安徽理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285;R96

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本文編號：2068758