蛋白激酶A参与抗胆碱药物快速抗抑郁作用机制研究

发布时间：2018-07-03 09:52

本文选题：东莨菪碱 + mTOR　；参考：《广州医科大学》2017年博士论文

【摘要】：研究背景:抑郁症严影响着着人类的健康,并给公共卫生事业带来巨大的负担。目前抑郁症的发病机制仍不十分明确,治疗现状并不理想。当前临床经典抗抑郁药多针对单胺类神经递质系统进行调控来发挥作用。这类药物普遍起效缓慢,往往需要数周乃至数月才见效,且疗效不佳,并可能增加患者自杀、致残的风险。因此,开发新型快速的抗抑郁药物具有着十分重要的临床和社会意义。近年来,研究发现,在亚临床剂量下,非选择性M型胆碱能受体阻断剂东莨菪碱具有快速而显著的抗抑郁作用,但其具体作用机制尚不明确。相关研究发现,与其他具有快速抗抑郁作用的药物类似,东莨菪碱通过增强AMPA受体功能,一过性地激活m TOR通路,进而重塑慢性应激导致的突触传递障碍。然而东莨菪碱究竟通过何种机制增强AMPA受体的功能,以及自阻断M受体到激活m TOR信号通路之间的胞内事件尚不明确。对这个问题的研究和阐明,将有助于揭示抗胆碱类药物快速抗抑郁的作用机制,并为开发新型快速安全的抗抑郁药物奠定理论基础。目的:本研究通过电生理技术,脑立体定位手术,急性海马脑片孵育,细胞膜蛋白提取,Western blot,习得性无助(Learned helplessness,LH)抑郁模型以及抑郁样表现的行为学检测等方法,探讨蛋白激酶A参与东莨菪碱对AMPA受体的增强以及激活m TOR通路的上游机制,并比较东莨菪碱与其他特异性M受体阻断剂的作用效果,为揭示快速抗抑郁药物作用机制和开发新型快速安全的抗抑郁药物提供重要的实验依据。方法:1、采用大鼠习得性无助(learned helplessness,LH)应激方法建立抑郁模型,并通过相关行为学实验(强迫游泳,开放旷场,糖水偏爱)检测造模效度。2、分别用0,10n M,100n M,1μM,10μM浓度东莨菪碱溶液灌流应激后大鼠脑片,检测东莨菪碱促进Glu A1 Ser845及m TOR Ser2448位点的磷酸化水平的作用与其浓度的关系。3、利用电生理技术,在NMDA受体的阻断剂APV孵育并灌流海马脑片的基础上,给予东莨菪碱(1μM)灌流,记录CA1辐射区兴奋性突触后场电位(SC-CA1 f EPSP)的变化,并以PKA的选择性的阻断剂H89(1μM)预孵育并灌流脑片,观察其对东莨菪碱这一作用的影响。4、急性分离的海马脑片予以东莨菪碱或合并H89进行孵育,用细胞膜蛋白提取技术分离膜蛋白进行Western blot检测,研究东莨菪碱及H89对Glu A1和Glu A2膜表达水平的影响。5、在经过GABAa受体阻断剂picrotoxin(100μM)预处理的海马脑片上,再给予东莨菪孵育,观察CA1区Glu A1 Ser845和m TOR Ser2448的磷酸化和Glu A1表达的增加作用是否被GABAa阻断剂所完全掩盖。6、通过立体定位置管手术,习得性无助大鼠侧脑室提前注射H89或人工脑脊液,然后腹腔注射东莨菪碱,或侧脑室注射M2受体选择性阻断剂美索拉明,取海马CA1区进行western blotting检测,观察东莨菪碱活体给药对Glu A1总蛋白,以及Glu A1 Ser845和m TOR Ser2448磷酸化水平的作用,以及H89预处理对该作用的影响。7、同上,侧脑室提前注射H89或人工脑脊液,再注射M2受体选择性阻断剂美索拉明,进行western blotting检测,观察其对海马Glu A1总蛋白,以及Glu A1 Ser845和m TOR Ser2448磷酸化水平的作用,以及H89预处理对该作用的影响。8、习得性无助大鼠侧脑室提前注射H89或人工脑脊液,随后给予东莨菪碱和美索拉明,进行强迫游泳和开放旷场检测,验证其活体条件下对抑郁样行为的作用,以及该效应是否受H89影响。结果:1、通过不可逃避足底点击应激,成功构建了SD大鼠习得性无助模型。2、急性海马脑片上,东莨菪碱剂量依赖地增加海马CA1区Glu A1 Ser845的磷酸化和Glu A1总蛋白的表达。东莨菪碱在1μM的浓度下增加m TOR Ser2448的磷酸化;在其他浓度则未见明显激活。3、东莨菪碱在APV(100μM)孵育和灌流下,依然能够快速增加海马SC-CA1兴奋性突触后场电位的幅度。这一效应可被蛋白激酶A的特异性拮抗H89阻断。4、东莨菪碱可以快速增加海马脑片CA1区胞膜Glu A1表达,该作用可以被H89所阻断。Glu A2膜表达则未受东莨菪碱和H89明显影响。5、GABAa阻断剂不能掩盖东莨菪碱介导的Glu A1 Ser845和m TOR Ser2448位点的磷酸化增加。但经过GABAa阻断剂预处理的脑片上,未能观察到东莨菪碱进一步增加Glu A1总蛋白的水平。6、LH大鼠腹腔注射东莨菪碱可以增加海马Glu A1总蛋白水平;这一效应不依赖于PKA。同时,东莨菪碱促进Glu A1 Ser845和m TOR Ser2448位点的磷酸化。该效应需要PKA途径参与。7、LH大鼠侧脑室注射美索拉明可以同时增加海马Glu A1 Ser845和m TOR Ser2448位点的磷酸化。该效应需要PKA途径参与。但经美索拉明和H89急性给药后,未观察到Glu A1总蛋白水平发生明显变化。8、LH大鼠腹腔注射东莨菪碱或侧脑室注射美索拉明都可以缩短强迫游泳实验中的不动时间,而不影响旷场试验中的自主活动能力。结论:1、PKA对于在东莨菪碱激活AMPA受体和m TOR通路,及发挥快速抗抑郁作用是必须的,其可能是通过磷酸化Glu A1 Ser845,促进AMPA受体上膜实现上述作用。2、东莨菪碱介导的Glu A1总蛋白快速表达增加不依赖PKA,但受GABAa受体调控。3、选择性M2受体阻断剂美索拉明可以起到类似于东莨菪碱对于Glu A1和m TOR的磷酸化作用,以及快速抗抑郁效果,这些作用依赖于PKA通路。这提示,对M2受体的抑制作用可能参与东莨菪碱激活PKA以及后继信号通路的机制。4、与东莨菪碱不同,美索拉明不影响Glu A1总蛋白表达。提示单独的M2-PKA机制短时间内不足以促进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.
【学位授予单位】：广州医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.4

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