烟碱受体拮抗剂α-芋螺多肽Eb1．6对L5脊神经切断大鼠热痛阈及脊髓星形胶质细胞和IL-1β表达水平的影响

Effects of nAChR antagonist α-conotoxin Eb1.6 on thermal pain threshold and spinal astrocytes activation and IL-β expression in rats using L_5 nerve transaction model

目的探讨烟碱受体拮抗剂旷芋螺多肽（α—conotoxin，α-CTX）Ebl．6对L5脊神经切断（SNT）大鼠热痛阈（TWL）及脊髓IL-1β表达水平和星形胶质细胞活性的影响。方法180～220g成年雄性SD大鼠50只，随机分为五组：假手术组（N组），SNT后分别腹腔注射α-CTXEb1．60．15nmol／kg组（E1组）、1．5nmol／kg组（E2组）、15nmol／kg组（E3组）和SNT后生理盐水对照组（C组），每组10只。在造模手术术后第7天疼痛模型稳定后开始腹腔注射相应剂量药物或生理盐水，连续用药7d。分别于术后第7天、术后第13天测定各组大鼠给药前、给药后1、2、4、7、12h热缩爪潜伏期（TWL）。于术后第13天行为学测试结束后将各组大鼠处死取L5脊髓组织，以Western blot法测定脊髓星形胶质细胞标志物胶质纤维酸性蛋白（glial fibrillary Acidic protein，GFAP）表达水平，ELISA法测定脊髓IL-1β表达水平。结果与给药前比较，E1、E2、E3组大鼠第7天给药后1、2、4hTWL明显延长（P〈0．05），给药后2h延长最明显；E1、E2、E3组大鼠第13天给药后1、2、4、7h以及E2、E3组第13天给药后12hTWL明显延长（P〈0．05），且均长于各组第7天相应时点的TWL（P〈0．05），给药后2h延长最明显；与E3组比较，E1、E2组大鼠第7天及第13天给药后2h的TWL明显缩短（P〈0．05）。且E1组明显短于E2组（P〈0．05）。与N组比较，E1、E2、E3组和C组大鼠脊髓IL-1β与GFAP表达水平明显升高（P〈0．05）；与C组比较，E1、E2、E3组脊髓IL-1β与GFAP表达水平明显下降（P〈0．05）；与E3组比较，E1、E2组脊髓IL-1β与GFAP表达水平明显升高（P〈0．05），且E1组明显高于E2组（P〈0．05）。结论腹腔注射Eb1.6能够剂量依赖性地减轻L5脊神经切断大鼠的热痛敏且连续用药能够增强其镇痛效果。其镇痛作用可能与抑制脊髓背角星形胶质细胞活化、降低IL-1β表达水平有一定关系。

Objective To observe the effects of nAChR antagonist α-conotoxin Eb1. 6 on thermal pain threshold and spinal IL-1β expression levels and astrocytes activation in rats using L5 spinal nerve transaction （SNT） model. Methods Fifty male Sprague-Dawley rats were randomly assigned into 5 groups with each group 10 rats： sham group, different doses of α-CTX Ebl. 6 （0. 15,1.5 and 15 nmol/kg） groups and the saline group after SNT. Saline solution or different doses of Ebl. 6 were intraperitoneally injected seven days after the surgery when the model was stable and the treatment continued for seven days. Measured the TWLs of all groups of the rats 1,2,4,7,12 hours after the injection on 7 d and 13 d. The rats were sacrificed and L5 spinal cord tissues were collected immediately after the behavioral tests on 13 d. The expression of GFAP and IL-1β were assessed by Western blot assay and enzyme linked immunosorbent assay （ELISA） separately. Results Groups E1, E2, E3 and C had shorter TWL before the injection on 7 d and 13 d than group N（P〈0.05）. The TWLs of the rats in groups E1,E2 and E3 of 1 h,2 h and 4 h after the injection on 7 d were significantly higher than that before the injection（P〈0.05） with 2 h after the injection showed the most obvious change. The TWL of 1 h,2 h,4 h and 7 h after the injection of the rats in group E1,E2 and E3 and those of 12 h after the injection of the rats in group E2 and E3 on 13 d were significantly higher than that before the injection（P〈0.05）and also higher than TWL of the respective time points on 7 d（P〈0. 05）, also with 2 h after the injection showed the most obvious change. The TWLs of 2 h after the injection among group E1,E2 and E3 showed significant differences both on 7 d and 13 d （P〈0. 05）. Rats spinal IL-1β and GFAP expression levels of group E1, E2, E3 and C were significantly higher than those of group N（P〈0.05）. Rats spinal IL-1β and GFAP expression levels of groups E1, E2, E3 significantly decreased compared with group C（P〈0. 05）. There Were significant differences among the spinal IL-1β and GFAP expression levels of group E1, E2 and E3 （P〈0.05）. Conclusion Ebl. 6 dose-dependently reduced the thermal hyperalgesia induced by L5 spinal nerve transection. Repeated treatment of Ebl. 6 could produce better analgesic effect, which might be partly attribute to the inhibition of spinal IL-β levels and astrocytes activation.