Neu-P11改善阿尔茨海默病模型大鼠认知功能损伤

发布时间：2019-05-29 05:33

【摘要】：目的：褪黑素是由5-羟色胺衍生而来一种吲哚类神经内分泌激素，主要由哺乳动物的松果体在夜间合成和分泌。近年来研究结果表明,外源性褪黑素可参与认知功能调节，并通过多种途径减轻AD的病理改变。Neu-P11是一种人工合成的新型褪黑素受体激动剂，具有高褪黑素受体亲和力及增强γ-氨基丁酸能神经传递的特性。本研究首先检测了Neu-P11能否增强物体识别记忆，然后基于两种AD大鼠模型检测了Neu-P11能否改善AD大鼠的认知功能损伤，最后基于PC12细胞模型对Neu-P11的作用机制进行了初步探讨。方法： 1、Neu-P11对大鼠物体识别记忆的影响：大鼠物体识别测试包括两个阶段即4分钟的训练期和3分钟的检测期。分别在上午或下午于训练期前2h腹腔注射溶媒、褪黑素或Neu-P11。训练结束后4h或24h，分别检测大鼠的短时程与长时程记忆保持。记录并分析各组大鼠在训练期的总探究时间与测试期的辨别指数。 2、Neu-P11对海马内微注射Aβ1-42诱导的AD模型大鼠认知功能损伤的影响：海马内微注射Aβ1-42诱导产生AD大鼠模型，建模后第21天与30天分别进行Y迷宫与物体识别记忆测试；行为测试完成后将大鼠断头取脑，行组织学检测。 3、Neu-P11对后扣带皮层内微注射叠氮化钠（NaN3）诱导早期AD模型大鼠认知功能损伤的影响：大鼠跳台被动回避测试包括训练与检测两个阶段。训练后1h将大鼠麻醉，进行后扣带皮层内NaN3微注射诱导早期AD大鼠模型。NaN3微注射后2h和6h分别进行一次Neu-P11及溶媒腹腔注射。跳台检测阶段于训练结束后24h进行，检测阶段结束后2h和4h分别行自发活动及高架十字迷宫测试以评估大鼠的活动能力与焦虑水平。行为测试完成后将大鼠断头取脑，行组织学检测。 4、Neu-P11对谷氨酸诱导PC12细胞损伤的影响：PC12细胞接种后24h，同时给予谷氨酸与Neu-P11或褪黑素处理；共孵育后24h，MTT法检测细胞活力，流式细胞仪检测细胞线粒体膜电位。 5、褪黑素受体拮抗剂Luzindole及5-HT1A受体拮抗剂WAY-100635对Neu-P11保护谷氨酸诱导PC12细胞损伤的影响：PC12细胞接种后24h，同时给予谷氨酸与Neu-P11处理；Luzindole和WAY-100635分别在Neu-P11处理的前30分钟和前10分钟加入到培养基，共孵育后24h，MTT法检测细胞活力，，流式细胞仪检测细胞线粒体膜电位。结果： 1. Neu-P11对大鼠物体识别记忆的影响：在短时及长时记忆测试中，无论上午或下午给药，Neu-P11处理组大鼠其辨别指数均显著性高于溶媒组，而褪黑素处理组大鼠仅在下午给药时其辨别指数显著性高于溶媒组。在各种处理条件下，各组大鼠在训练阶段对物体的总探究时间均无显著性组间差异。 2. Neu-P11对海马内微注射Aβ1-42诱导的AD模型大鼠认知功能损伤的影响：在Y迷宫测试中，各组大鼠总入臂次数无显著性组间差异。AD模型组大鼠交替比率显著性低于溶媒对照组，Neu-P11处理组大鼠交替比率显著性高于AD模型组大鼠，而褪黑素处理组大鼠交替比率与AD模型组大鼠比较无显著性组间差异。在物体识别测试中，各组大鼠在训练阶段对物体的总探究时间无显著性组间差异；在检测阶段，AD模型组大鼠辨别指数显著性低于溶媒对照组，Neu-P11处理组大鼠辨别指数显著性高于AD模型组大鼠，而褪黑素处理组大鼠辨别指数与AD模型组大鼠比较无显著性组间差异。 3. Neu-P11对后扣带皮层内微注射NaN3诱导早期AD模型大鼠认知功能损伤的影响：在跳台被动回避测试中，各组大鼠在训练阶段其跳台潜伏期无显著性组间差异；在检测阶段，溶媒对照组和Neu-P11对照组大鼠的跳台潜伏期显著性高于NaN3模型组大鼠；NaN3模型组大鼠其跳台潜伏期在训练与检测阶段无显著性差异；Neu-P11处理组大鼠在检测阶段的跳台潜伏期显著性高于训练阶段。此外，各组大鼠的活动距离及高架十字迷宫测试指标(开臂滞留时间百分比、开臂进入次数百分比及闭臂进入次数)无显著性组间差异。 4. Neu-P11对谷氨酸诱导PC12细胞损伤的影响：4mM谷氨酸可显著性诱导PC12细胞的活力与线粒体膜电位水平降低；Neu-P11与褪黑素处理可显著性改善谷氨酸诱导的损伤效应。 5. Luzindole及WAY-100635对Neu-P11保护谷氨酸诱导PC12细胞损伤的影响：4mM谷氨酸可显著性诱导PC12细胞的活力与线粒体膜电位水平降低；Neu-P11处理可显著性改善谷氨酸诱导的损伤效应。单独Luzindole或WAY-100635处理或共处理均阻断Neu-P11对谷氨酸诱导PC12细胞损伤的保护作用。结论： Neu-P11能够增强大鼠物体识别记忆改善AD模型大鼠认知功能损伤，Neu-P11对AD的改善作用可能与通过激活褪黑素与5-HT1A受体进而拮抗谷氨酸诱导的神经毒性相关。
[Abstract]:Purpose: Melatonin is a kind of neuroendocrine hormone derived from 5-hydroxytryptamine, which is synthesized and divided by the pineal body of the mammal at night. In recent years, the results show that the exogenous melatonin can be involved in the regulation of cognitive function, and the pathological changes of AD can be reduced by various means. Change. Neu-P11 is a synthetic novel melatonin receptor agonist, which has a high melatonin receptor affinity and a T-aminobutyric acid capable of neurotransmission. This study first examined whether Neu-P11 can enhance the recognition and memory of the object, and then, based on the two AD rat models, it is possible to improve the cognitive function damage of AD rats. Finally, the mechanism of the mechanism of Neu-P11 based on PC12 cell model is discussed. Please. Methods:1. The effect of Neu-P11 on the identification and memory of the rats: The identification and testing of the object in the rat consisted of two phases:4-minute training period and 3-minute training period. The detection period of the clock. The vehicle, melatonin or Ne were injected intraperitoneally at the morning or afternoon in the first 2 hours of the training period, respectively. U-P11. The short-term and long-term results of the rats were detected at 4 h or 24 h after the end of the training. The total study time and test period of each group of rats during the training period were recorded and analyzed. The effect of Nu-P11 on the cognitive function of rats with AD model induced by micro-injection of A-1-42 in the hippocampus: A model of AD rats induced by microinjection of A-1-42 in the hippocampus was induced by microinjection of A-1-42 in the hippocampus, and the Y-maze and the substance were respectively carried out on the 21 and 30 days after the modeling. body recognition and memory test; after the behavior test is finished, the rat is decapitated and taken into the brain The effect of NaN3 on the cognitive function of the rats with early AD model induced by NaN3 was studied by histological examination. The passive avoidance test of rats in rats was included. The training and detection were carried out in two stages. After the training, the rats were anesthetized and the NaN3 micro-injection in the cortex of the back buckle was induced. The model of early AD rats was established. Neu-P was performed at 2 h and 6 h after NaN3 microinjection. 11 and the vehicle were injected intraperitoneally. The stage of the stage detection was performed at 24 hours after the end of the training. The spontaneous activity and the elevated cross-maze test were performed at 2 and 4 hours after the end of the detection stage to evaluate the rats. Activity and anxiety levels. Rat decapitation after completion of the behavioral test The effects of 4, Neu-P11 on the injury of PC12 cells induced by glutamate were studied. The results showed that the cells were treated with glutamate and Neu-P11 or melatonin at 24 h after the inoculation of PC12 cells. The cell viability and flow cytometry were detected by MTT method after 24 h incubation. The effects of cell mitochondrial membrane potential,5, melatonin receptor antagonist Luzindole and 5-HT1A receptor antagonist WAY-100635 on the protective glutamate-induced PC12 cell injury were investigated by means of the instrument:24 h after the PC12 cell inoculation, and the glutamate and the Neu-P11 treatment at the same time; Luzindole and WAY-100635 were added to the first 30 minutes and the first 10 minutes of the Neu-P11 treatment, respectively. The cells were cultured for 24 h after incubation, and the cell viability and flow were measured by MTT method. Cytometer Detection of mitochondrial membrane potential of the cell. Results:1. The effect of 1. Neu-P11 on the identification and memory of an object in a rat: in a short and long-time memory test, the Neu-P11 treatment, whether administered in the morning or afternoon, In group rats, the discrimination index was significantly higher than that of the vehicle group, while the melatonin treatment group rats were only The differentiation index of the rats was significantly higher than that of the vehicle group at noon. Under various treatment conditions, the rats in each group were in the training stage. The effect of 2. Neu-P11 on the cognitive function of rats with AD model induced by micro-injection of A-1-42 in the hippocampus: in the Y-maze test There was no significant difference in the number of the total arms of the rats in the group. The alternating ratio of the rats in the AD model group was significantly lower than that of the vehicle control group, and the alternating ratio of the Neu-P11 treated group was significantly higher than that of the AD model group, while the alternating ratio of the melatonin treatment group to the rats There was no significant inter-group difference between the rate and the AD model group. In the object identification test, there was no significant difference in the total exploratory time of each group during the training phase; in the detection stage, the difference between the rats in the AD model group was significantly lower than that of the vehicle control group, and the Neu-P11 treatment The differentiation index of the rats in the group was significantly higher than that of the AD model group. No significant group difference between the number and the AD model group.3. The effect of neu-P11 on the cognitive function of the rats with early AD model induced by micro-injection of NaN3 in the back-buckled cortex: in the passive avoidance test of the stage, the rats in each group were in the training stage. There was no significant difference between the latency of the stage and the latency of the stage. In the detection stage, the latency of the jumping stage in the vehicle control group and the Neu-P11 control group was significantly higher than that of the NaN3 model group. The latency of the jumping stage in the NaN3 model group did not differ significantly between the training and the detection stage; the Neu-P11 treatment group was large. The incubation period of the rat in the detection stage was significantly higher than that of the training stage. In addition, the activity distance of each group and the elevated cross-maze test index (percentage of the open-arm retention time, the open-arm entry, No significant group difference was found between the number of times and the number of closed arms.4. The effect of Nu-P11 on the injury of PC12 cells induced by glutamate:4 mM glutamate significantly induced a decrease in the viability of PC12 cells and the level of mitochondrial membrane potential; Neu-P1 5. The effects of 5. Luzindole and WAY-100635 on the protective glutamate-induced PC12 cell injury were significantly improved by the treatment of melatonin.5. Luzindole and WAY-100635 had a significant effect on the protective glutamate-induced PC12 cell injury:4 mM glutamate significantly induced the decrease in the viability of PC12 cells and the level of mitochondrial membrane potential. The Neu-P11 treatment significantly improved glutamate-induced damage effects. Both the individual Luzindole or the WAY-100635 treatment or co-treatment block N eu- Conclusion: Neu-P11 can enhance the recognition and memory of the rat, improve the cognitive function of AD model, and the effect of Neu-P11 on AD may be related to the injury of PC12 cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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