氯胺酮介导的海马Neuroligin-1下调对创伤后应激功能障碍的影响

发布时间：2018-05-12 02:20

  本文选题：创伤后应激功能障碍 + 认知功能障碍　； 参考：《南京大学》2017年硕士论文

【摘要】：目的术中知晓是常见的全身麻醉术中并发症,其可导致患者产生严重的神经精神疾病,例如创伤后应激功能障碍(Post-traumatic stress disorder,PTSD)。PTSD主要表现为对创伤经历的再体验、逃避创伤相关事件和人、高应激三大核心症状,使患者承受沉重的神经精神负担。目前PTSD的具体病理生理机制尚不明确,因此有关于PTSD的研究属于当前研究热点。神经连接蛋白(Neuroligin-1,NLGN-1)主要表达于兴奋性突触上,胞内可通过突触后密度蛋白-95(Postsynaptic density protein 95,PSD-95)调控 N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR),羟基-5-甲基-4-异恶唑丙酸受体(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor,AMPAR)的表达,具有调控兴奋性突触形成及兴奋性突触信号传递的作用,与大脑认知功能有密切联系。NLGN-1参与自闭症、阿尔兹海默病(Alzheimer' s disease,AD)等认知障碍相关疾病的病理生理过程。NLGN1也可能与PTSD的情绪、学习障碍有关,但具体机制未明。氯胺酮(Ketamine,KET)是非竞争性的NMDAR拮抗剂,具有抗焦虑、抗抑郁的作用,有望成为新的PTSD治疗药物。本研究中,以NLGN-1为切入点,运用动物行为学及分子生物学探讨PTSD可能的病理生理机制及KET对其的作用机制,为PTSD的防治提供新的思路。方法雄性Sprague-Dawley大鼠(SD大鼠)250-275 g,通过足底电击建立PTSD动物模型。第一批SD大鼠随机均分为2组:对照组(Control组)、PTSD组。分别在建模后第7、14天通过条件性恐惧实验、水迷宫实验检测大鼠的恐惧记忆以及学习记忆能力;取前额叶皮层、海马、杏仁核,进行免疫印迹(Western blotting,WB)检测NLGN-1蛋白水平。第二批SD大鼠随机均分为4组:对照+生理盐水组(Control + NS 组)、对照 +KET 组(Control + KET 组)、PTSD+ 生理盐水组(PTSD+NS组)、PTSO + KET组(PTSO + KET组)。建模30min后腹腔注射KET 2.5 mg/kg,对照组给予同等体积的生理盐水,连续腹腔注射14天。于建模后第14天通过条件性恐惧实验、水迷宫实验检测大鼠的恐惧记忆以及学习记忆能力;取海马,进行WB检测NLGN-1、轴突蛋白-1(Neurexin-1,NRXN-1)、PSD-95、NMDAR-2B、AMPA-GluR1 蛋白表达水平。结果第一批实验中,无论是在建模后第7天,还是第14天,在条件性恐惧实验中,与Control组相比,PTSD组的僵直反应时间占总时间百分比均显著增加(P0.01);在水迷宫实验训练阶段,与Control组相比,训练第2、3、4天PTSD组的逃避潜伏期都明显延长(P0.05);在水迷宫实验测试阶段,与Control组相比,PTSD组的探索目标象限时间均无显著性差异(P0.05);在WB检测中,前额叶皮层、海马、杏仁核的NLGN-1在实验第7天并无改变(P0.05);在实验第14天,与Control组相比,PTSD组的前额叶皮层及杏仁核的NLGN-1蛋白表达水平无显著差异,但海马的NLGN-1蛋白水平表达升高(P0.05)。第二批实验中,条件性恐惧实验,与PTSD+NS组相比,PTSD+ KET组的僵直反应时间占总时间百分比显著减少(P0.01);水迷宫实验训练阶段,与PTSD+NS组相比,训练第2、4、5天PTSD + KET组的逃避潜伏期都明显缩短(P0.05);水迷宫实验测试阶段,各组的探索目标象限时间并无显著性差异(P0.05);在WB检测中,与PTSD + NS组相比,PTSD + KET组海马的NLGN-1、NRXN-1、PSD-95、NMDAR-2B、AMPA-GluR1蛋白表达水平都明显降低(P0.05)。结论PTSD大鼠存在恐惧记忆的增强及海马相关的空间学习能力的损伤,同时伴有海马的NLGN-1表达升高。给予KET可明显改善PTSD大鼠的恐惧记忆及海马相关的空间学习障碍,同时降低海马NLGN-1信号通路分子表达。本研究结果表明KET可能是通过降低NRXN-1-NLGN-1-PSD-95通路表达而抑制海马过度兴奋性发挥治疗作用的。
[Abstract]:Objective intraoperative knowledge is a common complication in general anesthesia, which can lead to severe neuropsychiatric disorders, such as post traumatic stress dysfunction (Post-traumatic stress disorder, PTSD).PTSD, which is mainly manifested in the re experience of traumatic experience, escaping from traumatic events and people, three core symptoms of high stress, and making the patient bear. At present, the specific pathophysiological mechanism of PTSD is not clear, so the research on PTSD is the current research hotspot. Neuroligin-1 (NLGN-1) is mainly expressed in the excitatory synapse, and the intracellular can be regulated by the postsynaptic density protein -95 (Postsynaptic density protein 95, PSD-95) to regulate the N- a. The expression of -D- aspartic acid receptor (N-methyl-D-aspartate receptor, NMDAR) and hydroxyl -5- methyl -4- isooxazole propionic acid receptor (alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPAR), which regulates excitatory synaptic formation and excitatory synaptic transmission, is closely associated with cognitive function of the brain. Participation in the pathophysiological processes of cognitive impairment related diseases such as autism, Alzheimer's disease, AD, and.NLGN1 may also be related to the mood of PTSD and learning disabilities, but the specific mechanism is not clear. Ketamine (KET) is a non competitive NMDAR antagonist with anti anxiety and antidepressant effects and is expected to become a new PTSD treatment. In this study, in this study, using NLGN-1 as the breakthrough point, the possible pathophysiological mechanism of PTSD and the mechanism of KET on it were explored by using animal behavior and molecular biology to provide new ideas for the prevention and control of PTSD. Methods male Sprague-Dawley rats (SD rats) were 250-275 g, and the animal model of PTSD was established by foot shock. The first batch of SD rats were random. The 2 groups were divided into 2 groups: the control group (group Control) and the PTSD group. The fear memory and learning memory ability of the rats were detected by the conditioned fear experiment on day 7,14 after the modeling. The prefrontal cortex, hippocampus, amygdala, Western blotting, WB were used to detect the level of NLGN-1 protein. The second batch of SD rats were randomly divided into two groups. 4 groups: control + physiological saline group (Control + NS group), control group +KET (Control + KET group), PTSD+ physiological saline group (PTSD+NS group), PTSO + KET group (PTSO + KET group). After modeling 30min, the abdominal injection was 2.5, the control group was given the same volume of saline, continuous intraperitoneal injection for 14 days. After the modeling, the conditioned fear experiment was passed fourteenth days after modeling, The water maze test was used to detect the memory and learning memory ability of rats; take the hippocampus, detect NLGN-1, -1 (Neurexin-1, NRXN-1), PSD-95, NMDAR-2B, AMPA-GluR1 protein expression by WB. Results in the first batch of experiments, seventh days after modeling, or fourteenth days, compared with the Control group, PTSD, PTSD, PTSD, compared with the Control group, PTSD. In the water maze test training stage, the escape latency of group PTSD was significantly longer than that of group Control (P0.05) in the training stage of the water maze test (P0.05), and there was no significant difference in the quadrant time between the PTSD group and the PTSD group in the water maze test stage, compared with the Control group (P0.05). In the WB test, the NLGN-1 of the prefrontal cortex, the hippocampus and the amygdala did not change at seventh days (P0.05). On the fourteenth day of the experiment, there was no significant difference in the level of NLGN-1 protein expression in the prefrontal cortex and amygdala of the PTSD group, but the level of NLGN-1 protein in the hippocampus increased (P0.05). The second batch of experiments showed that the conditioned fear was true. Compared with the PTSD+NS group, the PTSD+ KET group had a significant reduction in the total time percentage of the total time (P0.01), and the escape latency of the PTSD + KET group was significantly shortened (P0.05) in the training phase of the PTSD+NS group compared with the PTSD+NS group, and there was no significant difference in the quadrant time of the exploration targets in the water maze test stage. (P0.05); in the WB test, the expression level of NLGN-1, NRXN-1, PSD-95, NMDAR-2B, AMPA-GluR1 protein in the hippocampus of PTSD + KET group was significantly lower than that of the PTSD + NS group (P0.05). The fear memory of good PTSD rats and the spatial learning disorders related to the hippocampus also reduce the molecular expression of NLGN-1 signaling pathway in the hippocampus. The results of this study suggest that KET may play a therapeutic role in inhibiting hyperexcitability of the hippocampus by reducing the expression of the NRXN-1-NLGN-1-PSD-95 pathway.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.5
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本文编号：1876695