【作者】 周吉银；

【导师】 莫志贤；

【作者基本信息】 第一军医大学 ， 中药学， 2005， 硕士

【摘要】 背景:苯丙胺类中枢兴奋剂流行性滥用造成了严重的社会危害,对其干预是防治药物依赖的一个新课题。对苯丙胺依赖形成机制研究有待进一步深入。中药钩藤已在许多戒毒复方中使用,但其主要活性成分钩藤碱对苯丙胺类依赖的影响及作用机制尚未明确。 目的:复制苯丙胺依赖大鼠模型,观察钩藤碱对苯丙胺依赖大鼠条件性位置偏爱效应的影响,检测大鼠脑内伏核及杏仁核NMDA受体2B亚单位的表达及中枢氨基酸类、单胺类及内啡肽类神经递质含量的变化,探讨苯丙胺依赖形成的神经机制及钩藤碱抗苯丙胺依赖的作用机理。 设计:完全随机对照实验研究。 动物:实验动物采用雄性SD大鼠,经自然位置偏爱时间测定合格动物共96只,体重(200-260)g,由第一军医大学实验动物中心提供。随机分为空白对照组、苯丙胺模型组、阳性药氯胺酮组、钩藤碱系列剂量组及钩藤碱+生理盐水组。方法:钩藤碱对苯丙胺依赖大鼠条件性位置偏爱效应的影响及大鼠脑内伏核和杏仁核NMDA受体2B亚单位表达的实验共分7个组:即(1)空白对照组,(2)苯丙胺模型组,(3)氯胺酮(15mg/kg)+苯丙胺组,(4)钩藤碱低剂量(10mg/kg)+苯丙胺组,(5)钩藤碱中剂量(20mg/kg)+苯丙胺组,(6)钩藤碱高剂量(60mg/kg)+苯丙胺组,(7)钩藤碱(60mg/kg)+生理盐水组,每组8只大鼠。经基线测定合格后随机分组。动物给药方法如下:(2)-(6)组每天上午(8:00)给予苯丙胺(2mg/kg,sc),连续给药4d;(3)组从第3天起在给苯丙胺之前15min,ip给予氯胺酮(15mg/kg),连续3d;(4)-(6)组于第二天20:00(即给苯丙胺后12h),分别按上述剂量ip给予更多还原

【Abstract】 Background: The abuse of amphetamine type stimulants (ATS) have severely threaten to the human health and society. It is a new task to interfere with ATS dependence, but the mechanism of ATS dependence is not clear as yet. Ramulus Uncariae cum Uncis, a Chinese herbal medicine, has been used for drug dependence therapy. However, the effects and mechanism of rhychophylline, an active component, remain unknown.Objective: To establish amphetamine-dependent rat model and to explore the effects of rhychophylline on amphetamine-induced conditioned place preference (CPP) in rats. To examine the expressions of NMDAR 2B subunit in nucleus accumbens and amygdaloid and to detect central neurotransmitter levels in rat brain. To probe into the mechanisms of amphetamine dependence and effects of rhynchophylline on it. Design: A completely randomized controlled trial study.Animals: Male Sprague-Dawley rats, weighing 200-260g, obtained from experimental animal center of the First Military Medical University. 96 rats were selected and randomly divided into normal control group, amphetamine-dependent model group, ketamine (15 mg/kg) group, three dose groups of rhynchophylline and rhychophylline (60 mg/kg)+saline group.Methods: To study the effect of rhynchophylline on amphetamine-dependent conditioned place preference in rats and on NR2B, NR2B mRNA expressions innucleus accumbens and amygdaloid of rat brain, 56 rats were randomly divided into 7 groups (?=8): (l)normal control group, (2)amphetamine-dependent model group, (3)ketamine group, (4)rhynchophylline high dose (60 mg/kg) group, (5)rhynchophylline middle dose (20 mg/kg) group, (6)rhychophylline low dose (10 mg/kg) group, (7)rhychophylline+saline group. Each group rats were treated as follows: (2)-(6)groups were administered amphetamine(2 mg/kg, sc) at am 8:00 each day for 4 days; (3)group were injected of ketamine 15 min before administered amphetamine at d 3 for 3 days; (4)-(6)groups were treated with rhynchophylline (ip) respectively 12 h after injection of amphetamine at 2 d for 3 days; (l)group were administered saline instead of amphetamine and (7)group were administered (ip) rhynchophylline instead of amphetamine. All groups were given saline (0.5 ml, sc) at pm 16:00 each day for 4 days. Each group rats were placed in the amphetamine-paired compartment after administered drug in the morning and in the saline-pair compartment after treated with saline in the afternoon both for 1 h at 1 d for 4 days. The time of staying in the amphetamine-pair compartment in 15 min of each rat was measured 24 h after the last injection of amphetamine. After examination of CPP, all rats were anaesthetized with pentobarbital sodium. After intraventricle fixation with 4% paraformaldehyde, the rat brain was taken out, dehydrated and embedded in paraffin. In comparison with rat dissection picture, section which contained nucleus accumbens or amygdaloid was selected respectively. Immunohistochemistry (IH) and in situ hybridization (ISH) reaction were done according to operation procedure of Boster Corporation, each group had negative control test. Section was enveloped and photoed where contained nucleus accumbens or amygdaloid and the positive cells were taken count of under light microscope. Another 40 rats were randomly divided into 5 groups (n=8): rhynchophylline only had 60 mg/kg dose group, other groups were the same as former, so did treatment. After the time staying in amphetamine-paired compartment was measured, each rat was inspected with Encephalofluogram technology (ET)under a waked state for 18 min. The experiment data were statistically analyzed with SPSS 10.0 software and expressed as Mean±SD, one-way ANOVA and t test were used.Main outcome measures: The time staying of rats in the amphetamine-paired compartment for 15 min, distribution and numerical density of NR2B positive cells and NR2B mRNA expression in nucleus accumbens or amygdaloid, brain electrical activity maps and central neurotransmitter contents in brain.Results: After treated with amphetamine continuously for 4 days, the time staying in the amphetamine-paired compartment of model group rats was significantly longer than that of normal control group (P<0.01), which indicated that the rats had produced obvious conditioned place preference effect and amphetamine-dependent model was successfully established. The time of rhynchophylline middle and high doses groups wasn’t different from that of normal control group, the time of low dose group was a little longer than that of normal control group (P<0.05), but all obviously shorter than that of model group (P<0.01). The effect of rhynchophylline was increased in dose-dependence means. Rhynchophylline itself couldn’t induce a conditioned place preference in rats. The results of immunohistochemistry and in situ hybridization reaction indicated as follows: NR2B, NR2B mRNA positive cells lied both in cell membrane and cell plasm. The numerical density of NR2B and NR2B mRNA positive cells in nucleus accumbens and amygdaloid of model group was significantly higher than that of the other 6 groups (P<0.01). The numerical density of NR2B, NR2B mRNA positive cells of normal control group wasn’t different from that of ketamine group, rhynchophylline middle, high doses group and rhynchophylline+saline group, but obviously lower than that of rhynchophylline low dose group (P<0.0l). None of the four negative control tests had NR2B or NR2B mRNA positive cells in nucleus accumbens or amygdaloid. Brain electrical activity maps of normal control group appeared as "brain function balance picture", but that of model group showed almost disrupted brain function state, while that of ketaminegroup, rhynchophylline group and rhynchophylline+saline group were similar to that of normal control group. In comparison with normal control group, ketamine group, rhynchophylline group and rhynchophylline+saline group, IAAs, ACh and endorphin contents in brain of model group rats was obviously lower (P<0.01), on the contrary, EAAs, DA and NE level significantly higher (P<O.Ol); EAAs, ACh, DA, NE and endorphin contents of normal control group were not different from that of ketamine group, rhynchophylline group and rhynchophylline+saline group. 5-HT content of each group had no difference among each other.Conclusion: Rhynchophylline and ketamine could suppress acquisition of conditioned place preference in rats induced by amphetamine. Rhynchophylline itself didn’t show psychological effect in rats. Amphetamine could increase expressions of NR2B, NR.2B mRNA in nucleus accumbens and amygdaloid which might have something to do with the acquisition of amphetamine-dependent place preference. Middle, high doses of rhynchophylline and ketamine could obviously decrease NR2B, NR2B mRNA expressions induced by amphetamine, low dose of rhynchophylline could not effect their expressions. The suppression of rhynchophylline and ketamine on NR2B, NR2B mRNA expressions was similar to that of on acquisition of conditioned place preference. Amphetamine altered balance brain function of normal rat and distinctly disturbed central neurotransmitter levels. Rhynchophylline and ketamine could renew the disrupted brain function to normal state and markedly improve the abnormal state of neurotransmitter in amphetamine dependence rats.更多还原