脊髓ERK—CREB信号通路在吗啡依赖大鼠戒断反应中的作用

Roles of ERK-CREB signaling pathway in spinal cord in naloxone-induced withdrawal response in morphine-dependent rats

目的评价脊髓背角神经元细胞外信号调节激酶-cAMP反应元件结合蛋白（ERK-CREB）信号通路在吗啡依赖大鼠戒断反应中的作用。方法健康雄性SD大鼠，体重200～250g，2月龄。经枕骨大孔行鞘内置管，取置管成功的50只大鼠，采用随机数字表法，将其随机分为5组（n=10）：对照组（C组）、吗啡依赖组（MD组）、吗啡戒断组（MW组）、U0126组和二甲基亚砜组（DMSO组）。皮下注射吗啡10mg／kg，2次／d，隔天每次增加10mg／kg，至第6天末次注射50mg／kg，建立吗啡依赖模型。末次注射后4h，MW组、U0126组和DMSO组腹腔注射纳洛酮激发吗啡戒断反应。给予纳洛酮前30min时，U0126组和DMSO组分别鞘内注射U0126（溶于10ulDMSO中）150btg和DMSO10ul。于注射纳洛酮后1h内行戒断反应评分和促诱发痛评分，注射纳洛酮后1h处死大鼠，取脊髓组织，分别采用免疫组织化学法和Westernblot法测定脊髓背角磷酸化ERK（p-ERK）和磷酸化CREB（p-CREB）的表达。结果与c组比较，MW组脊髓背角p-ERK和p-CREB表达上调（P〈0．05）；与MD组比较，MW组、U0126组和DMSO组戒断反应评分和促诱发痛评分升高（P〈0．05）；与MW组比较，U0126组戒断反应评分和促诱发痛评分降低，脊髓背角p-ERK和p-CREB表达下调（P〈0．05），DMSO组上述指标差异无统计学意义（P〉0．05）。结论脊髓背角神经元ERK—CREB信号通路参与了吗啡依赖大鼠的戒断反应。

Objective To evaluate the role of extracelluar signal-regnlated kinase （ERK）-cyclic AMP response element binding protein （CREB） signaling pathway in the spinal cord in naloxone-induced withdrawal response in morphine-dependent rats. Methods Fifty male aduh SD rats, aged 2 months, weighing 200-250 g, in which intrathecal catheters were successfully implanted without complications, were randomly divided into 5 groups （n = 10 each）： group control （group C）; group morphine dependence （group MD）; group morphine withdrawal （group MW）; group U0126 （ERK signaling pathway blocker）; group dimethyl sulfoxide （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 6th day in groups MD, MW, U0126 and DMSO. Morphine withdrawal response was induced by intraperitoneal nal- oxone 4 mg/kg at 4 h after last morphine administration in groups MW, U0126 and DMSO. U0126 150 gg （in DM- SO 10ul ） and DMSO 10ul were administered intrathecally at 30 min before naloxone administration in groups U0126 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 phosphorylated ERK （p-ERK） and phosphorylated CREB （p-CREB） by immuno-histochemistry and Western blot. Results Morphine withdrawal significantly up-regulated the p-ERK and p-CREB expression in group MW compared with group C （ P 〈 0.05）. Withdrawal response score and touch evoked agitation score were significantly increased in groups MW, U0126 and DMSO as compared with group MD （P 〈 0.05）. U0126 pretreatment significantly attenuated naloxone-induced increase in withdrawal response score and touch evoked agitation score and down-regulated p-ERK and p-CREB expression in group U0126 as compared with group MW （P 〈 0.05）. Conclusion ERK-CREB signaling pathway in the spinal cord is involved in morphine withdrawal response in morphine-dependent rats.