氨甲蝶呤对大鼠疼痛行为及脊髓白细胞介素-6表达的影响

Effect of methotrexate on behavior of allodynia and spinal interleukin-6 expression

目的 观察鞘内注射免疫抑制性药物氨甲蝶呤对大鼠坐骨神经慢性结扎损伤（CCI）异常疼痛行为及脊髓白细胞介素-6（IL-6）表达的影响,探讨细胞因子的作用以及免疫途径治疗慢性病理性疼痛的可能性.方法 大鼠随机分为4组,每组10只：假损伤组,手术暴露大鼠左侧的坐骨神经但未予结扎,鞘内注射生理盐水;假治疗组,手术暴露大鼠左侧的坐骨神经但未予结扎,鞘内注射氨甲蝶呤;损伤组,制作CCI模型,鞘内注射生理盐水;治疗组,制作CCI模型,鞘内注射氨甲蝶呤.各组大鼠在手术前及术后的第2、5天鞘内注射氨甲蝶呤（1 mg/kg）或相当容积的生理盐水,在实验前和手术后第7天进行左足机械性痛觉异常和冷痛觉异常测定,并最后处死大鼠取脊髓标本,进行脊髓IL-6 mRNA表达的测定.结果 诱发损伤组大鼠左足缩腿反射的von-Frey毛序号在手术后（6.30±0.95）较实验前（12.00±0.67）减小（P＜0.05）,而治疗组手术后的von-Frey毛序号（9.30 ±0.95）虽比实验前（12.00 ±0.82）减小（P＜0.05）,但比损伤组有明显增加（P＜0.05）.在丙酮诱发的缩腿、抬足等反应的比率方面,手术后的损伤组（78.00±17.51）％和治疗组（36.00±15.78）％明显比实验前[（4.00±8.43）％和（6.00±9.66）％]高（P＜0.05）,但治疗组较损伤组有明显改善（P＜0.05）.损伤组大鼠脊髓IL-6 mRNA表达（0.80±0.11）比假损伤组（0.01±0.02）和假治疗组（0.01±0.02）明显增多（P＜0.05）,而治疗组IL-6 mRNA虽也有一定表达（0.44±0.09）,但与损伤组比较有明显减少（P＜0.05）.结论 脊髓中枢的IL-6可能在大鼠坐骨神经结扎损伤后的持续异常疼痛行为中起重要作用,氨甲蝶呤中枢给药可以抑制IL-6的作用并缓解大鼠的异常疼痛.

Objective To study the effect of methotrexate,the medicine of immunosuppressive action,on the behavior of allodynia and spinal interleukin （IL）-6 expression in the rat model of chronic ligation injury （CCI）,and then to explore the action of the cytokine and the possibility of the immune treatment to the chronic neuropathic pain.Methods The rats were randomly divided into four groups,10 rats for each group.In sham injuried group,the rats were operated to exposure the left sciatic nerves but not ligated,and the drug of intrathecal injection was saedline.In sham treated group,the rats were operated to exposure the left sciatic nerves but not ligated,and the drug of intrathecal injection was methotrexate.In injuried group,the rat CCI models were prepared,and were given saline intrathecal injection.In treated group,the rat CCI models were prepared,and the rats were given intrathecal injection of methotrexate.Before and 2 and 5 days after surgery,every rat was given intrathecal injection of methotrexate （1 mg/kg） or saline,which content was corresponded to that of methotrexate according to the weight of the rat.Before and 7 days post-operation,the behavior data of mechanical allodynia and cold allodynia were measured,and at end of the experiment,all rats were disposed and their spinal cords were harvested for determination of interleukin （IL）-6 mRNA expression.Results The serial number of von-Frey hair,which was used to bring out withdrawal responses,was （6.30 ± 0.95） after operation in the injured group,less than that before operation （12.00 ± 0.67,P ＜ 0.05）.In the treated group,the serial number of von-Frey hair after operation （9.30 ±0.95） was lower than that before surgery （12.00 ±0.82,P ＜0.05）.The serial number of yon-Frey hair after operation in the treated group was significantly increased as compared with that in the injuried group （P ＜ 0.05）.The ratio of acetone induced leg withdrawal and lifted reaction in the injuried group （78.00 ± 17.51） ％ and the treated group （36.00 ± 15.78） ％ after surgery was significantly higher than that before surgery [（4.00 ±8.43）％ and （6.00 ±9.66）％,P ＜0.05],but that in the treated group was significantly increased as compared with the injuried group （P ＜0.05）.The IL-6 mRNA expression in the injuried group （0.80 ±0.11） was significantly higher than in the sham injuried group （0.01 ± 0.02） and sham treated group （0.01 ±0.02,P ＜0.05）.The IL-6 mRNA expression was detectable in the treated group （0.44 ± 0.09）,but significantly reduced as compared with the injured group （P ＜ 0.05）.Conclusion After the ligated injury of the sciatic nerve of the rats,IL-6 may play an important role in the persistence of neuropathic pain in the spinal cord.The intrathecal injection of methotrexate may restrain the action of IL-6 and weaken the degree of allodynia in the rats.