鞘内注射NR2B反义寡核苷酸对纳洛酮诱发吗啡依赖大鼠戒断反应的影响

目的 评价鞘内注射N-甲基-D-天门冬氨酸(NMDA)受体2B亚基(NR2B)反义寡核苷酸对纳洛酮诱发吗啡依赖大鼠戒断反应的影响.方法 健康雌性SD大鼠,体重230～270 g,腹腔注射戊巴比妥钠60mg/kg麻醉下,于L3,4间隙穿刺置管.取鞘内置管成功的大鼠32只,随机分为4组(n=8):对照组(C组)、吗啡依赖组(MD组)、反义寡核苷酸组(AO组)和正义寡核苷酸组(SO组).MD组、AO组和SO组皮下注射吗啡10 mg/kg,2次/d,隔天增加10 mg/kg,至第6天末次注射50 mg/kg,建立吗啡依赖模型;C组皮下注射等量生理盐水.AO组和SO组于注射吗啡的同时分别鞘内注射15 nmol NR2B的反义寡核苷酸和正义寡核苷酸,用生理盐水稀释至5μl;C组和MD组鞘内注射等量生理盐水.各组在末次注射吗啡或生理盐水后4 h时,腹腔注射纳洛酮4 mg/kg诱发吗啡戒断.给予纳洛酮后30 min内观察戒断反应,并进行评分.计算大鼠体重变化幅度.给予纳洛酮后1 h时处死大鼠,测定海马NR1、NR2A和NR2B的mRNA表达水平.结果 与C组比较,MD组、AO组和SO组戒断反应评分和体重变化幅度升高,MD组和SO组海马组织NR2B mRNA表达上调(P<0.05或0.01);与MD组比较,AO组戒断反应评分和体重变化幅度降低,海马组织NR2A mBNA表达上调,NR2B mRNA表达下调(P<0.05或0.01),SO组各指标差异无统计学意义(P>0.05).各组海马组织NR1 mRNA表达比较差异无统计学意义(P>0.05).结论 鞘内注射NR2B反义寡核苷酸可抑制纳洛酮诱发吗啡依赖大鼠戒断反应,其机制与海马NMDA受体亚基水平和构成的变化调节有关.

Effect of NR2B antisense oligonucleotide on naloxone-induced withdrawal responses in morphine-dependent rats

Objective To investigate the effect of NR2B antisense oligonucleotide on naloxone-induced withdrawal responses in morphine-dependent rats. Methods Famale SD rats weighing 230-270 g were anesthetized with intraperitoneal pentobarhital 60 mg/kg. Intrathecal (IT)catheter was placed at L3,4 interspace.Thirty-two rats in which FT catheter was successfully placed were randomly divided into 4 groups ( n = 8 each) : group C control; group MD morphine dependence; group AO NR2B antisense oligonucleotide (aNR2B) and group SO NR2B sense oligonucleotide (sNR2B) . In group MD, AO, SO chronic morphine dependence was induced by increasing doses of subcutaneous morphine for 6 days. The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg twice every other day and reached 50 mg/kg on the 6th day. In group AO and SO IT aNR2B or sNR2B 15 nmol was administered simultaneously with subcutaneous morphine. Morphine withdrawal responses was induced by IT naloxone 4 mg/kg and scored based on the responses (0 = normal; higher scores signify severer responses) . The weight loss was calculated.The expression of NR1, NR2A and NR2B mRNA in hippocampus was determined by RT-PCR. Results The morphine withdrawal syndrome and weight loss were significantly incresed in group MD, AO and SO, while NR2B mRNA expression in hippocampus was up-regulated in group MD and SO compared with group C. The morphine withdrawal syndrome and weight loss were significantly decreased, NR2A mRNA expression in hippocampus was up-regulated and NR2B mRNA expression was down-regulated in group AO compared with group MD. There was no significant difference in NR1 mRNA expression between the 4 groups . Conclusion NR2B antisense oligonucleotide can suppress morphine withdrawal responses through the regulation of NMDA receptor level and construction in hippocampus.