本文選題：東莨菪堿 + mTOR�。� 參考：《廣州醫(yī)科大學(xué)》2017年博士論文

【摘要】：研究背景:抑郁癥嚴(yán)影響著著人類的健康,并給公共衛(wèi)生事業(yè)帶來巨大的負(fù)擔(dān)。目前抑郁癥的發(fā)病機(jī)制仍不十分明確,治療現(xiàn)狀并不理想。當(dāng)前臨床經(jīng)典抗抑郁藥多針對單胺類神經(jīng)遞質(zhì)系統(tǒng)進(jìn)行調(diào)控來發(fā)揮作用。這類藥物普遍起效緩慢,往往需要數(shù)周乃至數(shù)月才見效,且療效不佳,并可能增加患者自殺、致殘的風(fēng)險。因此,開發(fā)新型快速的抗抑郁藥物具有著十分重要的臨床和社會意義。近年來,研究發(fā)現(xiàn),在亞臨床劑量下,非選擇性M型膽堿能受體阻斷劑東莨菪堿具有快速而顯著的抗抑郁作用,但其具體作用機(jī)制尚不明確。相關(guān)研究發(fā)現(xiàn),與其他具有快速抗抑郁作用的藥物類似,東莨菪堿通過增強(qiáng)AMPA受體功能,一過性地激活m TOR通路,進(jìn)而重塑慢性應(yīng)激導(dǎo)致的突觸傳遞障礙。然而東莨菪堿究竟通過何種機(jī)制增強(qiáng)AMPA受體的功能,以及自阻斷M受體到激活m TOR信號通路之間的胞內(nèi)事件尚不明確。對這個問題的研究和闡明,將有助于揭示抗膽堿類藥物快速抗抑郁的作用機(jī)制,并為開發(fā)新型快速安全的抗抑郁藥物奠定理論基礎(chǔ)。目的:本研究通過電生理技術(shù),腦立體定位手術(shù),急性海馬腦片孵育,細(xì)胞膜蛋白提取,Western blot,習(xí)得性無助(Learned helplessness,LH)抑郁模型以及抑郁樣表現(xiàn)的行為學(xué)檢測等方法,探討蛋白激酶A參與東莨菪堿對AMPA受體的增強(qiáng)以及激活m TOR通路的上游機(jī)制,并比較東莨菪堿與其他特異性M受體阻斷劑的作用效果,為揭示快速抗抑郁藥物作用機(jī)制和開發(fā)新型快速安全的抗抑郁藥物提供重要的實(shí)驗(yàn)依據(jù)。方法:1、采用大鼠習(xí)得性無助(learned helplessness,LH)應(yīng)激方法建立抑郁模型,并通過相關(guān)行為學(xué)實(shí)驗(yàn)(強(qiáng)迫游泳,開放曠場,糖水偏愛)檢測造模效度。2、分別用0,10n M,100n M,1μM,10μM濃度東莨菪堿溶液灌流應(yīng)激后大鼠腦片,檢測東莨菪堿促進(jìn)Glu A1 Ser845及m TOR Ser2448位點(diǎn)的磷酸化水平的作用與其濃度的關(guān)系。3、利用電生理技術(shù),在NMDA受體的阻斷劑APV孵育并灌流海馬腦片的基礎(chǔ)上,給予東莨菪堿(1μM)灌流,記錄CA1輻射區(qū)興奮性突觸后場電位(SC-CA1 f EPSP)的變化,并以PKA的選擇性的阻斷劑H89(1μM)預(yù)孵育并灌流腦片,觀察其對東莨菪堿這一作用的影響。4、急性分離的海馬腦片予以東莨菪堿或合并H89進(jìn)行孵育,用細(xì)胞膜蛋白提取技術(shù)分離膜蛋白進(jìn)行Western blot檢測,研究東莨菪堿及H89對Glu A1和Glu A2膜表達(dá)水平的影響。5、在經(jīng)過GABAa受體阻斷劑picrotoxin(100μM)預(yù)處理的海馬腦片上,再給予東莨菪孵育,觀察CA1區(qū)Glu A1 Ser845和m TOR Ser2448的磷酸化和Glu A1表達(dá)的增加作用是否被GABAa阻斷劑所完全掩蓋。6、通過立體定位置管手術(shù),習(xí)得性無助大鼠側(cè)腦室提前注射H89或人工腦脊液,然后腹腔注射東莨菪堿,或側(cè)腦室注射M2受體選擇性阻斷劑美索拉明,取海馬CA1區(qū)進(jìn)行western blotting檢測,觀察東莨菪堿活體給藥對Glu A1總蛋白,以及Glu A1 Ser845和m TOR Ser2448磷酸化水平的作用,以及H89預(yù)處理對該作用的影響。7、同上,側(cè)腦室提前注射H89或人工腦脊液,再注射M2受體選擇性阻斷劑美索拉明,進(jìn)行western blotting檢測,觀察其對海馬Glu A1總蛋白,以及Glu A1 Ser845和m TOR Ser2448磷酸化水平的作用,以及H89預(yù)處理對該作用的影響。8、習(xí)得性無助大鼠側(cè)腦室提前注射H89或人工腦脊液,隨后給予東莨菪堿和美索拉明,進(jìn)行強(qiáng)迫游泳和開放曠場檢測,驗(yàn)證其活體條件下對抑郁樣行為的作用,以及該效應(yīng)是否受H89影響。結(jié)果:1、通過不可逃避足底點(diǎn)擊應(yīng)激,成功構(gòu)建了SD大鼠習(xí)得性無助模型。2、急性海馬腦片上,東莨菪堿劑量依賴地增加海馬CA1區(qū)Glu A1 Ser845的磷酸化和Glu A1總蛋白的表達(dá)。東莨菪堿在1μM的濃度下增加m TOR Ser2448的磷酸化;在其他濃度則未見明顯激活。3、東莨菪堿在APV(100μM)孵育和灌流下,依然能夠快速增加海馬SC-CA1興奮性突觸后場電位的幅度。這一效應(yīng)可被蛋白激酶A的特異性拮抗H89阻斷。4、東莨菪堿可以快速增加海馬腦片CA1區(qū)胞膜Glu A1表達(dá),該作用可以被H89所阻斷。Glu A2膜表達(dá)則未受東莨菪堿和H89明顯影響。5、GABAa阻斷劑不能掩蓋東莨菪堿介導(dǎo)的Glu A1 Ser845和m TOR Ser2448位點(diǎn)的磷酸化增加。但經(jīng)過GABAa阻斷劑預(yù)處理的腦片上,未能觀察到東莨菪堿進(jìn)一步增加Glu A1總蛋白的水平。6、LH大鼠腹腔注射東莨菪堿可以增加海馬Glu A1總蛋白水平;這一效應(yīng)不依賴于PKA。同時,東莨菪堿促進(jìn)Glu A1 Ser845和m TOR Ser2448位點(diǎn)的磷酸化。該效應(yīng)需要PKA途徑參與。7、LH大鼠側(cè)腦室注射美索拉明可以同時增加海馬Glu A1 Ser845和m TOR Ser2448位點(diǎn)的磷酸化。該效應(yīng)需要PKA途徑參與。但經(jīng)美索拉明和H89急性給藥后,未觀察到Glu A1總蛋白水平發(fā)生明顯變化。8、LH大鼠腹腔注射東莨菪堿或側(cè)腦室注射美索拉明都可以縮短強(qiáng)迫游泳實(shí)驗(yàn)中的不動時間,而不影響曠場試驗(yàn)中的自主活動能力。結(jié)論:1、PKA對于在東莨菪堿激活A(yù)MPA受體和m TOR通路,及發(fā)揮快速抗抑郁作用是必須的,其可能是通過磷酸化Glu A1 Ser845,促進(jìn)AMPA受體上膜實(shí)現(xiàn)上述作用。2、東莨菪堿介導(dǎo)的Glu A1總蛋白快速表達(dá)增加不依賴PKA,但受GABAa受體調(diào)控。3、選擇性M2受體阻斷劑美索拉明可以起到類似于東莨菪堿對于Glu A1和m TOR的磷酸化作用,以及快速抗抑郁效果,這些作用依賴于PKA通路。這提示,對M2受體的抑制作用可能參與東莨菪堿激活PKA以及后繼信號通路的機(jī)制。4、與東莨菪堿不同,美索拉明不影響Glu A1總蛋白表達(dá)。提示單獨(dú)的M2-PKA機(jī)制短時間內(nèi)不足以促進(jìn)Glu A1快速合成。
[Abstract]:Research background: depression affects human health and brings great burden to public health. The pathogenesis of depression is still not very clear and the treatment status is not ideal. The current clinical classic antidepressants are mainly regulated to the monoamine neurotransmitter system. These drugs are generally slow to take effect. It often takes weeks and even months to be effective, not effective, and may increase the risk of suicide and disability. Therefore, the development of a new fast antidepressant has a very important clinical and social significance. In recent years, the study found that the non selective M cholinergic receptor blocker, scopolamine, is fast at subclinical doses. Significant antidepressant effects, but the specific mechanisms of action are not clear. Related studies have found that scopolamine is similar to other drugs with rapid antidepressant effects, and scopolamine activates the AMPA receptor function, activates the m TOR pathway, and then reshape the contact disorder caused by chronic stress. The function of enhancing the AMPA receptor and the intracellular events from blocking the M receptor to the activation of the m TOR signaling pathway are not clear. The study and clarifying of this problem will help to reveal the mechanism of anti cholinergic antidepressant action and lay a theoretical foundation for the development of a new fast and safe antidepressant. Electrophysiological techniques, stereotaxic surgery, acute hippocampal slices incubation, cell membrane protein extraction, Western blot, Learned helplessness (LH) depression model and behavioral detection of depressive symptoms, and explore the protein kinase A participation in the enhancement of scopolamine and the upstream mechanism of the m TOR pathway. The effects of scopolamine and other specific M receptor blockers were compared to provide important experimental basis for revealing the mechanism of rapid antidepressant action and the development of new fast and safe antidepressants. Methods: 1, the model of depression was established by the learned helplessness (LH) stress method. In order to study the experiment (forced swimming, open field, sugar water preference), the model validity.2 was detected by using 0,10n M, 100N M, 1 u M, and 10 M concentration of scopolamine solution after irrigation of rat brain slices, and the relationship between the effect of scopolamine on the phosphorylation level of Glu A1 Ser845 and m TOR sites and its concentration were detected by electrophysiological technology. On the basis of the receptor blocker APV incubation and perfusion of hippocampal slices, scopolamine (1 mu M) was administered to record the changes in the excitatory postsynaptic potential (SC-CA1 f EPSP) of the excitatory synapse in the CA1 radiation area, and preincubated with the selective blocker of PKA, H89 (1 u M), to observe the effect of scopolamine on the effect of scopolamine,.4, the hippocampus of acute separation. The brain slices were incubated with scopolamine or H89, and the membrane protein extraction technique was used to separate the membrane protein for Western blot detection. The effect of scopolamine and H89 on the expression level of Glu A1 and Glu A2 membrane was studied. The increased effect of phosphorylation of Glu A1 Ser845 and m TOR Ser2448 on the expression of Glu A1 is completely concealed by GABAa blockers, through stereotaxic catheterization, and early injection of H89 or artificial cerebrospinal fluid in the acquired helplessness of the rat's lateral ventricles, then intraperitoneal injection of scopolamine, or lateral ventricle injecting the M2 receptor selective blocker of molamine, The effect of scopolamine on Glu A1 total protein, Glu A1 Ser845 and m TOR Ser2448 phosphorylation level, and the effect of H89 preconditioning on this effect were observed by Western blotting detection in CA1 region of hippocampus. Western blotting detection was used to observe its effect on the total protein of Glu A1 in the hippocampus, as well as the phosphorylation of Glu A1 Ser845 and m TOR Ser2448, and the effect of H89 pretreatment on this effect. Test the effect of the depressive behavior on depressive behavior and whether the effect was affected by H89. Results: 1, the acquisition of SD rat acquired helplessness model.2 was successfully constructed by inescaping foot click stress. On acute hippocampal slices, scopolamine increased the phosphorylation of Glu A1 Ser845 and the total Glu A1 protein of Glu A1 Ser845 in the hippocampus CA1 region. Scopolamine increased the phosphorylation of M TOR Ser2448 at the concentration of 1 u M; no.3 was activated in other concentrations. Scopolamine still could increase the amplitude of the excitatory postsynaptic potential of hippocampal SC-CA1 excitatory synapse rapidly under the incubation and perfusion of APV (100 M). This effect could be blocked by the specificity antagonistic H89 of the egg white kinase A, which blocked the.4, scopolamine. The expression of Glu A1 in the CA1 region of the hippocampal slices can be rapidly increased, which can be expressed by the.Glu A2 membrane blocked by H89, but not affected by scopolamine and H89, and GABAa blocking agents can not hide the increase of scopolamine mediated Glu A1 Ser845 and phosphorylation. It was found that scopolamine further increased the level of Glu A1 total protein level.6, and intraperitoneal injection of scopolamine in LH rats could increase the total protein level of Glu A1 in the hippocampus. This effect was not dependent on PKA., and scopolamine promoted the phosphorylation of Glu A1 Ser845 and m TOR. It can also increase the phosphorylation of Glu A1 Ser845 and m TOR Ser2448 site at the same time. This effect requires PKA pathway. However, the total protein level of Glu A1 has not been significantly changed after the acute administration of the drug, and the intraperitoneal injection of scopolamine in the LH rats or the injection of the lateral ventricle can shorten the forced swimming test. Conclusion: 1, PKA is necessary for the activation of the AMPA receptor and the m TOR pathway in scopolamine and the rapid antidepressant effect. It may be through the phosphorylation of Glu A1 Ser845 to promote AMPA receptor membrane to achieve the above effect.2, scopolamine mediated Glu A1 total protein fast. The increase in expression is not dependent on PKA, but by the GABAa receptor regulation of.3, the selective M2 receptor blocker may be similar to the phosphorylation of scopolamine to Glu A1 and m TOR, as well as the rapid antidepressant effect, which are dependent on the PKA pathway. This suggests that the inhibitory effect of the M2 receptor may be involved in the PKA and after the activation of scopolamine. The mechanism.4 of the signal pathway, unlike the scopolamine, does not affect the total protein expression of Glu A1, suggesting that a single M2-PKA mechanism is not sufficient to promote the rapid synthesis of Glu A1 in a short time.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R749.4

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