鞘内注射米诺环素对尼古丁戒断大鼠脊髓背角Iba-1和CX3C趋化因子受体1表达的影响

Effect of intrathecal injection of minocycline on expression of Iba-1 and CX3C chemokine receptor 1 proteins in spinal dorsal horn of rat with nicotine withdrawal

目的 观察鞘内注射小胶质细胞抑制剂米诺环素对尼古丁戒断大鼠脊髓背角CX3C趋化因子受体1（CX3C chemokine receptor 1, CX3CR1）表达的影响。 方法 健康雄性SD大鼠60只，体重180～250 g，将大鼠按随机数字表法分成5组（每组12只）：正常对照组（Normal组）、尼古丁组（NT组）、尼古丁注射前预先给药组（M＋NT组）、尼古丁戒断后给药组（NT＋M组）、尼古丁＋溶酶剂二甲基亚砜组（NT＋DMSO组）。Normal组不作任何处理；NT组、M＋NT组、NT＋M组和NT＋DMSO组连续7 d每天7：00，15：00和23：00皮下注射尼古丁9 mg·kg^-1·d^-1。末次注射后60 min皮下注射美加明1 mg/kg。NT＋M组和NT＋DMSO组在戒断后分别鞘内注射米诺环素50 g/10 L和二甲基亚砜10 μl，1次/d，连续3 d。M＋NT组于尼古丁开始注射前1 h，鞘内注射米诺环素5 g/L，以后每天给药直至戒断后3 d。观察大鼠尼古丁戒断后各组热缩足潜伏期（paw withdrawal latenc, PWL）的变化。戒断后第4 天每组各处死6只大鼠，取其L4～L6脊髓节段，采用免疫荧光法检测脊髓背角CX3CR1和Iba-1表达水平的变化；Western blot法检测CX3CR1蛋白的表达变化。 结果 与Normal组比较，M＋NT组PWL无明显变化（P〉0.05），脊髓背角Iba-1（小胶质细胞）和CX3CR1表达变化差异无统计学意义（P〉0.05）；与NT组比较，NT＋M组鞘内注射米诺环素第2天开始，大鼠的PWL明显增加（P〈0.05），但仍低于Normal组（P〈0.05）；与Normal组比较，NT组尼古丁戒断后第4天Iba-1表达显著增多（P〈0.05）；与NT组比较，NT＋M组Iba-1、CX3CR1的表达明显减少（P〈0.05），但表达量仍多于Normal组（P〈0.05）。 结论 鞘内注射米诺环素可以明显抑制尼古丁戒断大鼠的热痛敏，并引起脊髓背角Iba？蛳1和CX3CR1的表达变化，表明小胶质细胞和CX3CR1均参与了尼古丁戒断大鼠痛觉过敏的形成和发展，并提示CX3CR1可能作为小胶质细胞的下游机制参与了尼古丁戒断大鼠痛觉过敏的发生、发展过程。

Objective To investigate the effect of intrathecal injection of minocycline on expressions of CX3C chemokine receptor 1（CX3CR1） in spinal dorsal horn of rats with NT. Methods Sixty SD rats, were randomly divided into 5 groups（n=12）： normal group （Normal）, nicotine withdrawal group（NT）, pre-minocycline treated＋nicotine withdrawal group（M＋NT）, nicotine withdrawal＋post-minocycline treated group （NT＋M）, nicotine withdrawal＋dimethyl sulfoxide （DMSO） group （NT＋DMSO）. A rat model of NT was established by subcutaneous injection of pure nicotine（3 mg/kg）, three times daily for 7 d. Hyperalgesia was evaluated by observing the paw withdrawal latency（PWL）. The activation of microglia was investigated by immunofluorescence staining. The expression level of CX3CR1 was studied by Western blot and immunofluorescence staining. Results The PWL of rats in NT＋M group was significantly increased compared to NT group（P〈0.05）, but still lower than the Normal group. Compared to NT group, the expression levels of microglia and CX3CR1 in NT＋M group were significantly decreased（P〈0.05）, but still more than Normal group （P〈0.05）. Conclusions Intrathecal administration of the microglia specific inhibitor minocycline can attenuate thermal hyperalgesia induced by nicotine withdrawal. Activated microglia may upregulate the expression of CX3CR1 which contribute to the development of hyperalgesia following nicotine withdrawal.