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

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

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200-250g，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（cremo．phor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10ms／ks，2次，d，隔天每次增加10mg／kg，至第6天末次注射50ms／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、150126组、cremophor组和DMSO组腹腔注射纳洛酮4ms／kg激发吗啡戒断反应。给予纳洛酮前30min时，L-NAME组、7-Ni组、AG组、U0126组、cremophor组和DMSO组分别鞘内注射L广N．硝基精氨酸甲酯400／Lg（溶于10出生理盐水中）、7-硝基吲唑400btg（溶于10pl克列莫佛中）、氨基胍400tLg（溶于10出生理盐水中）、U0126150ttg（溶于10出二甲基亚砜中）、克列莫佛10肛l和二甲基亚砜10μl。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-Nj组、AG组、U0126组、DMS0组和cremophor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。

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 inhibi tor） （ 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 DblSO.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 μg, AG 400 ttg, U0126 150 μg, cremopher 10 μl and DMSO 10 μl were administered IT at 30 rain 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 compared with 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.