米诺环素抑制卫星胶质细胞激活对于三叉神经痛大鼠模型镇痛机制的研究

Analgesic mechanism of minocycline inhibiting activation of glial cells in rat models of trigeminal neuralgia

目的研究米诺环素通过抑制三叉神经痛大鼠三叉神经节卫星胶质细胞的炎症反应镇痛的机制，为临床三叉神经痛的治疗提供实验支持。方法（1）采用激光化学诱导三叉神经损伤构建三叉神经痛大鼠模型。将30只SD大鼠按照随机数字表法分为假手术组，模型组，米诺环素组（50μg米诺环素灌胃7d），每组10只。测定3组大鼠神经颜面部支配区机械痛阈值，检测大鼠三叉神经节内核转录因子-κB（NF-κB）、白介素（IL）—1β的蛋白和mRNA表达，胶质纤维酸性蛋白（GFAP）染色观察卫星胶质细胞的激活，同时行NF-κB免疫组织化学染色。（2）采用硝酸甘油构建卫星胶质细胞的炎症模型，体外培养SD大鼠三叉神经部位卫星胶质细胞，将细胞模型分为对照组、模型组、米诺环素低剂量组（15μmol／L）、高剂量组（30μmol／L），检测细胞内IL-1β和NF—κB的表达，Fluo-3／AM探针负载检测卫星胶质细胞中钙离子浓度的变化。结果（1）米诺环素组的疼痛阈值显著高于模型组，差异有统计学意义（P〈0．05）。米诺环素组中NF-κB、IL-1β的蛋白水平以及mRNA的表达水平相比模型组显著降低，差异均有统计学意义（P〈0．05）。NF—κB染色实验中，假手术组NF—κB染色呈弱阳性，模型组呈强阳性，米诺环素组表达较模型组明显减弱。GFAP染色实验中．米诺环素组阳性细胞数明显低于模型组，差异有统计学意义（P〈0．05）。（2）米诺环素组细胞IL-1β和NF-κB蛋白及mRNA表达均显著低于模型组，差异均有统计学意义（P〈0．05）。米诺环素组细胞内钙离子浓度明显降低，与模型组比较差异有统计学意义（P〈0．05）。结论米诺环素可以通过抑制卫星胶质细胞的激活，降低炎症因子NF-κB、IL-1β的水平来改善三叉神经痛。

Objective To study the mechanism of minocycline inhibiting inflammatory reaction of trigeminal ganglion glial cells in trigeminal neuralgia rats, and provide reference and support for treatment of trigeminal neuralgia. Methods （1） The rat models of trigeminal neuralgia were established by laser chemical induction of trigeminal nerve injury. Thirty SD rats were randomly divided into sham-operated group, model I group, minocycline group （intragastric administration of 50 p.g minocycline for 7 d, n=10）. The threshold of mechanical pain was measured in the facial nerve areas of rats. The protein and mRNA expressions of nuclear transcription factor （NF）-KB and interleukin （IL）-1β in the trigeminal ganglion were detected, the activation of satellite glial cells was observed by glial fibrillary acidic protein （GFAP） staining, and immunohistochemical staining was used to detect NF-κB level. （2） The inflammatory models ofglial cells were established with nitroglycerin; and trigeminal glial cells from the SD rats were cultured in vitro; the cell models were divided into control group, model Ⅱ group, low-dose minocycline group （15 μmol/L）, and high-dose minocycline group （30 μmol/L）; the expressions of NF-κB and IL-1β in cells were detected. Fluo-3/AM probe load was used to observe the concentration changes of calcium ions in the glial cells. Results （1） The threshold of pain intrigeminal neuralgia of minocycline group was significantly higher than that of model Ⅰ group （P〈0.05）; the protein and mRNA expressions of NF-κB and IL-1β in the minocycline group were significantly decreased as compared with those in model Ⅰ group （P〈0.05）; weak NF-κB expression was noted in the sham-operated group, strong NF-κB expression was noted in the model Ⅰ group, and that in the minocycline group was obviously decreased as compared with that in model Ⅰ group; number of GFAP positive cells in the minocycline group was significantly smaller as compared with the model Ⅰ group （P〈0.05）. （2）The protein and mRNA expressions of NF-κB and IL-1β in the low-dose minocycline group and high-dose minocycline group were significantly decreased as compared with those in the model Ⅱ group （P〈0.05）; and the concentration of calcium ions in astrocytes of the low-dose minocycline group and high-dose minoeyeline group was significantly decreased as compared with that of model Ⅱ group （P〈0.05）. Conclusion Minocycline can alleviate pain in trigeminal neuralgia rats by inhibiting the activation of satellite glial cells and decreasing the levels of inflammatory factors NF-κB and IL-1β.