氯胺酮对糖尿病神经痛合并抑郁大鼠海马GABA_B受体及BDNF表达的影响

发布时间：2018-05-21 13:11

本文选题：氯胺酮 + GABAB受体　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：糖尿病神经痛（diabetic neuropathy pain, DNP）是糖尿病常见的并发症之一，在糖尿病患者中的发病率约为7.5%-24%，且其发病率呈逐年上升的趋势，严重影响人们的生活质量和心理健康。抑郁作为一种常见的情绪障碍与慢性疼痛密切相关。目前，大约12.7%的糖尿病神经痛患者合并抑郁，但临床尚缺乏有效的治疗药物及措施。氯胺酮作为一种非竞争性的NMDA（N-甲基-D-天冬氨酸）受体拮抗剂，不仅具有显著的镇痛效果，近年来也发现其具有快速持久的抗抑郁作用，但其镇痛抗抑郁的具体机制尚不清楚，因此本研究通过其机制探讨为临床治疗提供理论依据。研究表明：丘脑、海马、大脑皮层等区域的GABAB（γ-氨基丁酸）受体蛋白表达下调在抑郁、焦虑、成瘾等的形成过程中发挥着重要的作用，并且发现糖尿病神经痛大鼠脊髓GABAB受体表达下调在疼痛的形成和维持中发挥重要作用。另有研究发现：在疼痛合并抑郁时，海马、丘脑、大脑皮层等区域神经元释放脑源性神经生长因子（BDNF）明显减少，给予镇痛抗抑郁治疗后，BDNF表达升高直至恢复正常，但在应用氯胺酮镇痛抗抑郁的过程中海马GABAB受体及BDNF表达的变化目前尚不清楚。本实验通过腹腔注射链脲佐菌素（STZ），制备糖尿病神经痛大鼠模型，应用强迫游泳实验制备并评价抑郁模型，糖尿病神经痛合并抑郁模型制备成功后，给予氯胺酮及NMDA受体激动剂NMDA进行干预。实验分为：正常对照组（C组）、糖尿病神经痛合并抑郁模型（D1组）、氯胺酮组（D2组）、NMDA组（D3组）、氯胺酮+NMDA组（D4组），通过观察不同组别大鼠50%机械缩足阈值（PWT）、强迫游泳实验不动时间（IMSFT）变化，采用免疫组织化学染色法、RT-PCR法及Western blot法，比较各组大鼠海马GABAB受体及BDNF的表达变化，从而探讨氯胺酮镇痛抗抑郁作用的可能机制。方法：健康成年雄性SD大鼠30只，体重180～200g，由河北医科大学实验动物中心提供，随机分为两组：正常对照组（C组）和糖尿病神经痛模型组（D组），腹腔分别注射生理盐水或链脲佐菌素(STZ，60mg/kg)。20只大鼠腹腔注射STZ两周后，有16只大鼠的空腹血糖16.7mmol/L，为糖尿病模型制备成功，然后对糖尿病大鼠进行15min强迫游泳实验（每周1次）制备抑郁模型，方法为：将大鼠置于高40cm，直径20cm圆柱形透明水缸中，水深25±5cm，水温25±2℃，水面距缸上缘10±5cm，水深以大鼠后爪刚可触及缸底却不足以支撑身体为宜，游泳15min。腹腔注射STZ三周后，用von Frey Hairs测定机械缩足阈值，计算50%机械缩足阈值小于4g为糖尿病神经痛大鼠模型制备成功(D组)。将正常对照组(C组)和实验组（D组）大鼠，通过5min强迫游泳实验记录其不动时间（immobility time of forced swimming test, IMFST：大鼠在水中停止挣扎、或显示漂浮状态、或仅作一些必要的轻微动作保持头部浮在水面上的时间视为不动时间）。每只大鼠进行强迫游泳实验之前更换玻璃缸内的水，洗净水缸，避免相互影响。与C组大鼠相比，IMFST显著延长者提示DNP合并抑郁模型可能制备成功。为验证模型制备成功，在行为学（PWT及IMFST）测定结束后，取C组和D组大鼠海马组织，应用免疫组化法测定BDNF阳性细胞数，分子生物学方法测定其mRNA和蛋白表达变化。结合行为学及分子生物学变化，确定DNP合并抑郁大鼠的行为学条件，用于下一步成功制备DNP合并抑郁大鼠模型。根据上一步实验结果确定的模型制备成功条件，制备DNP合并抑郁模型大鼠80只。根据给药不同随机分为四组（n=20）：模型组（D1组）：生理盐水（0.5ml）+生理盐水（0.5ml）；氯胺酮组（D2组）：氯胺酮10mg/kg(0.5ml)+生理盐水(0.5ml)；NMDA组（D3）：生理盐水(0.5ml)+NMDA15mg/kg(0.5ml)；氯胺酮+NMDA组（D4组）：氯胺酮10mg/kg(0.5ml)+NMDA15mg/kg(0.5ml)。连续肌肉注射给药一周，，每天上午给药。给药完成后第1天(T1)、给药完成后两周（T2）分别测定大鼠PWT及IMFST，并于T1、T2时间点行为学测试后每组分别取10只大鼠海马组织，采用免疫组化法和分子生物学技术测定大鼠海马GABAB受体及BDNF表达变化。结果：1腹腔注射STZ2周末，20只大鼠中有16只空腹血糖16.7mmol/L，为糖尿病模型制备成功。上述16只大鼠经过1次15min的强迫游泳实验，于STZ注射3周末测定PWT及IMFST，其中10只大鼠PWT＜4g为大鼠DNP模型成功，且其IMFST均＞150s，其余大鼠剔除实验。与正常大鼠相比，10只DNP组大鼠PWT明显降低, IMFST明显延长，其海马组织BDNF阳性细胞数明显减少；mRNA含量明显下降；蛋白表达显著下降（P0.05）。因此，当行为学测定糖尿病大鼠PWT＜4g且IMFST＞150s时，其海马组织BDNF表达明显下降，表明DNP合并抑郁模型制备成功。2与C组比较，D组大鼠各时间点PWT降低（P0.05），IMFST延长（P0.05），BDNF阳性细胞数减少（P0.05），mRNA及蛋白表达下降（P0.05），上述结果与模型大鼠变化一致，表明实验模型制备成功，同时GABAB受体蛋白、mRNA表达以及阳性细胞数均减少（P0.05）；与D1组比较，D2组大鼠海马BDNF及GABAB受体的蛋白及mRNA表达均增加，阳性细胞表达数也明显上升（P0.05）；而D3、D4组大鼠海马GABAB受体、BDNF的蛋白、mRNA表达以及阳性细胞数均未见明显变化（P0.05）。上述指标各组T1与T2时间点相比均无明显变化（P＞0.05）。结论：氯胺酮可通过上调糖尿病神经痛合并抑郁大鼠海马GABAB受体及BDNF的表达，改善大鼠疼痛及抑郁状态。
[Abstract]:Objective: diabetic neuropathy pain (DNP) is one of the common complications of diabetes. The incidence of diabetes is about 7.5%-24%, and its incidence is increasing year by year, which seriously affects people's quality of life and mental health. Depression as a common emotional disorder is closely related to chronic pain. At present, about 12.7% of patients with diabetic neuropathic pain are associated with depression, but there are still lack of effective therapeutic drugs and measures. Ketamine, as a non competitive NMDA (N- methyl -D- aspartic acid) receptor antagonist, not only has significant analgesic effect, but has also found its rapid and persistent antidepressant effect in recent years, but its analgesic and inhibitory effect The mechanism of depression is not clear. Therefore, this study will provide theoretical evidence for clinical treatment through its mechanism.
The study shows that the downregulation of GABAB (gamma aminobutyric acid) receptor protein expression in the thalamus, hippocampus and cerebral cortex plays an important role in the formation of depression, anxiety and addiction, and it is found that the down regulation of GABAB receptor expression in the spinal cord of diabetic rats plays an important role in the formation and maintenance of pain and pain. Now: the release of brain derived Neurogrowth factor (BDNF) in hippocampus, thalamus, cerebral cortex and other regions in pain and depression is significantly reduced. After the treatment of analgesic antidepressant treatment, the expression of BDNF increases until normal, but the changes in the expression of GABAB receptor and BDNF in hippocampus are not yet clear during the application of ketamine analgesic and antidepressant. The rat model of diabetic neuropathic pain was prepared by intraperitoneal injection of streptozotocin (STZ). Forced swimming test was used to prepare and evaluate the depression model. After the diabetic neuropain combined with depression model was successfully prepared, ketamine and NMDA receptor agonist NMDA were given. The experiment was divided into normal control group (C group) and diabetic neuralgia. Combined depression model (group D1), ketamine group (group D2), group NMDA (group D3) and group +NMDA (group D4) of ketamine (group D4), by observing the threshold of 50% mechanical contraction (PWT) in different groups of rats (PWT), the change of the time (IMSFT) of forced swimming test (IMSFT), immunohistochemistry, RT-PCR method and Western blot method were used to compare the hippocampal GABAB receptors and the tables of the rats in each group. To explore the possible mechanism of ketamine analgesia and antidepressant effect.
Methods: 30 healthy adult male SD rats, weighing 180 to 200g, were provided by the experimental animal center of Hebei Medical University. They were randomly divided into two groups: normal control group (group C) and diabetic neuropain model group (group D), peritoneal injection of saline or streptozotocin (STZ, 60mg/kg).20 in the abdominal cavity for two weeks, and 16 rats in the abdominal cavity. The fasting blood glucose 16.7mmol/L was successfully prepared for the diabetic model. Then the 15min forced swimming test (1 times a week) was used to prepare the depressive model. The method was to put the rats in a high 40cm, diameter 20cm cylindrical transparent cylinder, the water depth was 25 + 5cm, the water temperature was 25 + 2 C, the water surface was 10 + 5cm from the upper edge of the cylinder, and the water depth was just a palpable cylinder in the hind paw of the rat. The bottom was not sufficient to support the body. After three weeks of intraperitoneal injection of 15min. for swimming STZ, the threshold of mechanical contraction was measured with von Frey Hairs, and a successful model of diabetic neuropathic rat model was successfully prepared (group D) with the threshold of 50% mechanical shrinkage less than 4G. (group C) and experimental group (D group), the time of action was recorded by 5min forced swimming test (group D). Immobility time of forced swimming test, IMFST: the rats in the water stop struggling, or display the floating state, or only make some necessary minor movements to keep the head floating on the surface of the water as immobile). Each rat can replace the water in the glass cylinder before the forced swimming test, clean the water cylinder, avoid mutual influence. And C group. In order to verify the success of the DNP combined depression model, the IMFST significant prolongation suggested that the model was successful. In order to verify the success of the model preparation, the hippocampal tissues of the C and D groups were taken after the determination of the behavioral (PWT and IMFST). The number of BDNF positive cells was measured by immunohistochemical method, and the molecular biology method was used to determine the changes of mRNA and protein expression. And molecular biological changes, determine the behavioral conditions of DNP combined with depression rats, and prepare for the next successful preparation of DNP combined with depression rat model.
According to the successful condition of the model preparation determined by the results of the previous experiment, 80 rats with DNP combined with depression were prepared. According to the different dosage, the model group was randomly divided into four groups (n=20): the model group (group D1): physiological saline (0.5ml) + physiological saline (0.5ml); ketamine group (D2 group): chloramines 10mg/kg (0.5ml) + physiological saline (0.5ml); NMDA group (D3): physiological salt Water (0.5ml) +NMDA15mg/kg (0.5ml), ketamine +NMDA group (D4 group): Ketamine 10mg/kg (0.5ml) +NMDA15mg/kg (0.5ml). Continuous intramuscular injection for one week, every morning, after first days (T1). After the administration was completed, the rats were measured respectively, and 10 rats in each group were taken after the time point behavior test. The expression of GABAB receptor and BDNF in hippocampus of rats were determined by immunohistochemistry and molecular biology techniques.
Results: 1 on the weekend of intraperitoneal injection of STZ2, 20 rats had 16 fasting blood glucose 16.7mmol/L, which were successfully prepared for diabetes model. After 1 times of 15min forced swimming test, 16 rats were tested for PWT and IMFST at the end of STZ injection, of which 10 rats PWT < 4G as the DNP model of rats, and the IMFST > 150s, the rest of the rats were eliminated. Compared with normal rats, the PWT of 10 rats in the 10 group was obviously decreased, the IMFST was obviously prolonged, the number of BDNF positive cells in the hippocampus decreased obviously, the content of mRNA decreased obviously and the protein expression decreased significantly (P0.05). Therefore, the BDNF expression of the hippocampus was obviously decreased when the behavioral test of diabetic rats PWT < 4G and IMFST > 150s. Compared with group C, PWT decreased (P0.05), IMFST prolonged (P0.05), BDNF positive cells decreased (P0.05), mRNA and protein expression decreased (P0.05) at all time points in group D, and the above results were consistent with the model rats, indicating that the experimental model was successfully prepared, while the GABAB protein, the expression of positive cells and the number of positive cells were reduced. Less (P0.05); compared with group D1, the protein and mRNA expression of BDNF and GABAB receptors in hippocampus of group D2 increased, and the expression of positive cells also increased significantly (P0.05), while D3, BDNF protein, mRNA expression and number of positive cells were not obviously changed. Significant changes (P > 0.05).
Conclusion: ketamine can improve pain and depression in rats by up regulating the expression of GABAB receptor and BDNF in diabetic rats with neuropathic pain and depression.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】