脊髓NO信号通路和ERK信号通路在吗啡依赖大鼠戒断反应中的作用

目的 评价脊髓NO信号通路和细胞外调节激酶(ERK)信号通路在吗啡依赖大鼠戒断反应中的作用.方法 鞘内置管成功的健康雄性SD大鼠90只,体重200～ 250 g,采用随机数字表法,将其随机分为9组(n＝10):正常对照组(C组)、吗啡依赖组(MD组)、吗啡戒断组(MW组)、L-N-硝基精氨酸甲酯组(L-NAME组)、7-硝基吲唑组(7-Ni组)、氨基胍组(AG组)、U0126组、克列莫佛组(cremophor组)和二甲基亚砜组(DMSO组).MD组、MW组、L-NAME组、7-Ni组、AG组、U0126组、cremophor组和DMSO组皮下注射吗啡10 mg/kg,2次/d,隔天每次增加10 mg/kg,至第6天末次注射50 mg/kg,建立吗啡依赖模型.末次注射吗啡后4h时,MW组、L-NAME组、7-Ni组、AG组、U0126组、cremophor组和DMSO组腹腔注射纳洛酮4 mg/kg激发吗啡戒断反应.给予纳洛酮前30 min时,L-NAME组、7-Ni组、AG组、U0126组、cremophor组和DMSO组分别鞘内注射L-N-硝基精氨酸甲酯400μg(溶于10μl生理盐水中)、7-硝基吲唑400 μg(溶于10μl克列莫佛中)、氨基胍400μg(溶于10 μl生理盐水中)、U0126 150μg(溶于10μl二甲基亚砜中)、克列莫佛10 μl和二甲基亚砜10 μl.注射纳洛酮后1h内观察大鼠戒断反应和痛觉异常反应,并进行评分,然后处死大鼠,取脊髓组织,分别采用免疫组化法和Western blot 法测定脊髓背角诱导型一氧化氮合酶(iNOS)、神经型一氧化氮合酶(nNOS)和磷酸化ERK(p-ERK)的表达.结果 与MD组比较,MW组、L-NAME组、7-Ni组、AG组、U0126组、DMSO组和crmophor组戒断反应评分和促诱发痛评分升高(P<0.05)；与MW组比较,L-NAME组、7-Ni组、AG组和U0126组戒断反应评分和促诱发痛评分降低(P<0.05),DMSO组和cremophor组差异无统计学意义(P＞0.05)；与C组和MD组比较,MW组脊髓背角nNOS和iNOS表达上调(P<0.05)；与MW组比较,U0126组脊髓背角nNOS和iNOS表达下调(P<0.05).与C组比较,MD组和MW组脊髓背角p-ERK表达上调(P＜0.05)；与MW组比较,L-NAME组、7-Ni组和AG组脊髓背角p-ERK表达下调(P<0.05).结论 脊髓NO信号通路和ERK信号通路的相互调节作用参与了吗啡依赖大鼠的戒断反应.

Role of NO and ERK signaling pathways in spinal cord in naloxone-induced withdrawal response in morphine-dependent rats

Objective To investigate the role of NO and extracellular signal-regulated kinase (ERK) signaling pathways in the spinal cord in naloxone-induced withdrawal response in morphine-dependent rats.Methods Ninety male adult SD rats weighing 200-250 g in which IT catheters were successfully implanted without complication were randomly divided into 9 groups (n ＝ 10 each):group control (group C); group morphine dependence (group MD); group morphine withdrawal (group MW); group N(G)-nitro-L-arginine methyl ester (eNOS inhibitor) (L-NAME group) ; group 7-nitroindazole (nNOS inhibitor) (group 7-Ni) ; group aminoguanidine(iNOS inhibitor) (group AG); group U0126 (ERK signaling pathway blocker); group cremophor (solvent for 7-Ni) and group DMSO (solvent for U0126).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 until 50 mg/kg on the 6th day in groups MD,MW,L-NAME,7-Ni,AG,U0126,cremophor and DMSO.Morphine withdrawal response was induced by intraperitoneal (IP) naloxone 4 mg/kg at 4 h after last morphine administration in groups MW,L-NAME,7-Ni,AG,U0126,cremophor and DMSO.L-NAME 400 μg,7-Ni 400 μ g,AG 400 μg,U0126 150 μg,cremopher 10 μl and DMSO 10 μl were administered IT at 30 min before naloxone administration in groups L-NAME,7-Ni,AG,U0126,cremophor and DMSO respectively.Morphine withdrawal response (0 ＝ no withdrawal response,3 ＝ severe response) and touch evoked agitation (0 ＝ no agitation,2 ＝ severe agitation) were observed and scored during 1 h after naloxone administration.The animals were then sacrificed and the spinal cord was removed for determination of the expression of iNOS,nNOS and phosphor-ERK (p-ERK) by immunohisto-chemistry and Western blot.Results Morphine withdrawal significantly increased withdrawal response score and touch evoked agitation score in group MW as comparedwith group MD.L-NAME,7-Ni,AG and U0126 pretreatment significantly attenuated naloxone-induced increase in withdrawal response score and touch evoked agitation score in groups L-NAME,7-Ni,AG and U0126 as compared with group MW.Morphine withdrawal significantly up-regulated the nNOS and iNOS expression in group MW compared with groups C and MD.L-NAME,7-Ni and AG pretreatment significantly down-regulated p-ERK expression in groups L-NAME,7-Ni and AG as compared with group MW.Conclusion The interaction between NO and ERK signaling pathways may be involved in morphine withdrawal response in morphine-dependent rats.