TSPO选择性配体YL-IPA08的抗焦虑、抗抑郁作用机制研究

发布时间：2018-05-20 05:04

本文选题：18 + ku转位蛋白(TSPO)　；参考：《河北北方学院》2017年硕士论文

【摘要】：18 ku转位蛋白(Translocator protein,TSPO),曾被称为外周型苯二氮卓类受体,其在脑内主要位于胶质细胞线粒体外膜上,近年来被认为是焦虑症和抑郁症治疗的潜在新靶标。该蛋白的主要功能是介导胆固醇进入线粒体内膜,经过酶促反应合成神经类固醇(如四氢孕酮),进而通过作用于GABAA受体调节情绪行为。AC-5216是国际公认的TSPO高选择性配体,但具有不溶于水、生物利用度低等缺陷。YL-IPA08是军事医学科学院毒物药物研究所对AC-5216进行结构优化合成的新型配体化合物,具有TSPO亲和力和选择性更高、易溶于水,生物利用度高等优点。前期已证实YL-IPA08在啮齿动物实验上具有显著的抗焦虑、抗抑郁、抗PTSD作用,且脑内TSPO及其介导的四氢孕酮合成释放介导了上述行为活性。本研究利用斑马鱼行为模型和皮质酮(corticosterone,CORT)诱导BV-2胶质细胞系凋亡模型,探讨YL-IPA08抗焦虑、抗抑郁作用及其抗细胞凋亡机制,对解析TSPO的靶标价值及其配体药物作用机制具有重要意义。采用新鱼缸实验、黑白偏好实验、镜像实验这三个经典的斑马鱼行为模型,连续进行YL-IPA08药物处理一周于第八天进行行为学检测,探讨该药在斑马鱼模型上的行为学效应。采用高浓度CORT诱导BV-2细胞凋亡模型,以YL-IPA08和(或)TSPO拮抗剂PK11195预处理细胞2 h,而后与CORT共处理24 h,采用流式细胞技术检测细胞凋亡率,CCK-8法检测细胞活性,Western蛋白印迹法检测TSPO的表达,酶联免疫吸附试验(ELISA)检测细胞上清中四氢孕酮的水平。本研究结果如下:(1)与正常对照组相比,新鱼缸实验中,YL-IPA08 0.012mg·L~(-1)在不影响游动距离的情况下能够增加实验鱼开始6 min内在鱼箱中的上下穿梭次数和在鱼箱上半部分的停留时间(P0.05);黑白偏好实验中,YL-IPA08 0.012 mg·L~(-1)能够降低实验鱼开始6 min内在暗区的停留时间百分比(P0.05),同时增加其在明区的停留时间百分比(P0.05);镜像实验中,YL-IPA08 0.012 mg·L~(-1)能够减少实验鱼首6 min在鱼箱中央区的游动距离百分比和停留时间百分比(P0.05),但不减少进入中央区的次数;(2)采用流氏细胞仪进行细胞凋亡检测发现:与溶剂对照组相比,CORT诱导细胞凋亡实验中,100、200μmol·L~(-1)的CORT能够有效诱导BV-2细胞凋亡(P0.05,P0.01),且200μmol·L~(-1)时作用更为显著;YL-IPA08 1~100 nmol·L~(-1)与阳性化合物AC-5216一样能够显著降低CORT处理的BV-2细胞的凋亡率(P0.01),且具剂量依赖关系,该作用能够被TSPO拮抗剂PK11195 100 nmol·L~(-1)所拮抗(P0.05);CCK-8细胞活性检测显示,YL-IPA08 100 nmol·L~(-1)能够显著升高细胞活性(P0.01),PK11195 100 nmol·L~(-1)处理会拮抗该作用(P0.01);Western蛋白印迹法结果显示YL-IPA08 100 nmol·L~(-1)能够显著增加CORT处理的BV-2细胞TSPO蛋白的表达(P0.05),同时ELISA法检测显示YL-IPA08 100nmol·L~(-1)显著升高细胞培养液中四氢孕酮的水平(P0.01),PK11195 100nmol·L~(-1)显著逆转该现象,降低四氢孕酮的水平(P0.05)。以上结果提示,TSPO选择性配体YL-IPA08在斑马鱼行为模型上具有抗焦虑、抗抑郁行为作用,并且显著对抗高浓度皮质酮诱导的BV-2细胞凋亡,增加细胞活性,YL-IPA08的行为作用可能与其升高四氢孕酮水平并产生胶质细胞保护作用有关。
[Abstract]:18 Ku transposition protein (Translocator protein, TSPO), once known as the peripheral type of benzene two azeptors, is mainly located on the outer membrane of the mitochondria of the glial cells. In recent years, it has been considered as a potential new target for the treatment of anxiety and depression. The main function of this protein is to mediate cholesterol into the mitochondrial membrane and synthesize by enzymatic reaction. Neurosteroid (such as four hydro progesterone), and then through the action of the GABAA receptor to regulate emotional behavior.AC-5216 is an internationally recognized TSPO high selective ligand, but it is insoluble in water and low bioavailability.YL-IPA08 is a new ligand compound for the structure optimization of AC-5216 by the Toxicological Research Institute of Military Medical Science Academy of the PLA. TSPO has the advantages of high affinity and selectivity, easy to dissolve in water and high bioavailability. Earlier, it has been proved that YL-IPA08 has significant anti anxiety, antidepressant and anti PTSD effects in rodent experiments, and the synthesis and release of four hydrogen progesterone mediated by TSPO in the brain mediates the activity. This study uses the zebrafish behavior model and the cortex Corticosterone (CORT) induces the apoptosis model of BV-2 glial cell line, and discusses the anti anxiety, antidepressant and anti apoptosis mechanism of YL-IPA08. It is of great significance to the target value of TSPO and the mechanism of ligand drug action. The three classic zebrafish behavior models are used in the new fish tank experiment, black and white preference experiment and mirror image experiment. A week of YL-IPA08 drug treatment was performed on eighth days to investigate the behavioral effects of the drug on the zebrafish model. The apoptosis model of BV-2 cells was induced by high concentration of CORT. YL-IPA08 and (or) TSPO antagonist PK11195 were used to pretreat cell 2 h, and then CORT was treated with 24 h. Flow cytometry was used to detect cell apoptosis. Rate, CCK-8 method to detect cell activity, Western blot method to detect TSPO expression, enzyme linked immunosorbent assay (ELISA) to detect the level of four hydrogen progesterone in cell supernatant. The results of this study are as follows: (1) compared with normal control group, YL-IPA08 0.012mg. L ~ (-1) can increase the experimental fish without affecting the distance of swimming. At the beginning of the 6 min, the number of up and down in the fish box and the stop time (P0.05) in the upper part of the fish box (P0.05); in the black and white preference experiment, YL-IPA08 0.012 mg. L~ (-1) can reduce the percentage of residence time (P0.05) of the experimental fish in the dark area of the 6 min, and increase the percentage of the residence time in the clear area (P0.05); in the mirror experiment, YL-IPA08 0 .012 mg. L~ (-1) can reduce the percentage of swimming distance and the percentage of residence time (P0.05) in the central area of the fish box in the central area of the fish box, but it does not reduce the number of times into the central area; (2) the apoptosis detection by the flow cytometer is found: compared with the solvent control group, the CORT energy of 100200 Mu mol. L~ (-1) in the CORT induced apoptosis experiment. BV-2 cell apoptosis (P0.05, P0.01) can be effectively induced, and the effect of 200 mu mol. L~ (-1) is more significant; YL-IPA08 1~100 nmol L~ (-1) can significantly reduce the apoptosis rate of the cells treated with the positive compound AC-5216, and has a dose-dependent relationship. 5) CCK-8 cell activity detection showed that YL-IPA08 100 nmol / L~ (-1) could significantly increase cell activity (P0.01), PK11195 100 nmol. L~ (-1) could antagonize the action (P0.01). The results showed that YL-IPA08 100nmol. L~ (-1) significantly increased the level of four h progesterone in cell culture fluid (P0.01), PK11195 100nmol. L~ (-1) significantly reversed this phenomenon and reduced the level of four hydrogen progesterone (P0.05). The above results suggest that TSPO selective ligands have anti anxiety, antidepressant action and significant antagonism on the zebrafish behavior model. High concentration of corticosterone induced apoptosis of BV-2 cells and increased cell activity. The action of YL-IPA08 may be related to the increased level of four hydro progesterone and the protective effect of glial cells.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749

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