枢经推拿对神经病理性疼痛大鼠NLRP3炎症体活化的影响及镇痛机制研究

Effect of Shujing Tuina on Activation of NLRP3 Inflammation in Rats with Neuropathic Pain and Its Analgesic Mechanism

目的基于枢经学说探讨推拿对神经病理性疼痛(NPP)大鼠NLRP3炎症体活化的影响及其镇痛分子学机制。方法将40只SD大鼠随机分为5组:正常组、模型组、假手术组、推拿组、假推拿组,每组8只。除外正常组和假手术组,其余3组均建立L5脊神经结扎模型(SNL模型)。于术前(0 d)、术后第3天和第7天对各组大鼠测定其热痛缩足反应潜伏期(PWTL)和机械性刺激缩足阈值(PWMT);于术后第1天开始对假推拿组、推拿组进行干预,连续干预7 d,其余3组不予干预,观察7 d;于术后第7天运用Western blot技术检测大鼠L4-6脊髓背角NLRP3、ASC、Caspase-1蛋白的表达水平,运用ELISA技术检测血清IL-1β含量。结果术前各组大鼠PWTL和PWMT差异无统计学意义;术后第3天模型组、推拿组、假推拿组PWTL和PWMT较正常组、假手术组显著降低(P<0.01),模型组、推拿组、假推拿组之间的PWTL和PWMT差异无统计学意义;术后第7天推拿组PWTL和PWMT高于模型组和假推拿组(P<0.01)。术后第7天模型组NLRP3、ASC、Caspase-1及IL-1β水平较正常组、假手术组增高(P<0.01),与模型组、假推拿组相比,推拿组显著下调大鼠NLRP3、ASC、Caspase-1及IL-1β的表达水平(P<0.05)。结论推拿可减轻SNL模型大鼠痛敏反应,其机制与下调脊髓背角NLRP3、ASC、Caspase-1蛋白的表达,从而抑制NLRP3炎症体的活化,阻碍IL-1β成熟和分泌,阻断其介导的免疫炎症反应有关。

Objective To explore the effect of Tuina on the activation of NOD-like receptor protein 3(NLRP3)inflammasome in neuropathic pain(NPP)rats and its analgesic molecular mechanism based on the Shujing theory.Methods Forty SD rats were randomly divided into 5 groups:normal group,model group,sham operation group,Tuina group,and sham Tuina group,with 8 rats in each group.Except for the normal group and the sham operation group,the other 3 groups were established L5 spinal nerve ligation model(SNL model).Before operation(0 d)and on 3 rd and 7 th day after operation,the paw withdraw thermal latency(PWTL)and paw withdrawal mechanical threshold(PWMT)of rats in each group were tested.The sham Tuina group and the Tuina group were intervened on the 1 st day after surgery for 7 days continuously,while the other 3 groups were not intervened and observed for 7 days.Western blot was used to detect the expression levels of NLRP3,ASC and Caspase-1 protein in the dorsal horn of rats L4-6 spinal cord on the 7 th day after surgery.ELISA technology was used to detect the content of serum interleukin-1β(IL-1β).Results There was no statistically significant difference in PWTL or PWMT between rats in each group before operation.The levels of PWTL and PWMT in the model group,Tuina group and sham Tuina group were significantly lower than those in the normal group and sham operation group on the 3 rd day after operation(P<0.01),and there was no statistically significant difference among the model group,the Tuina group and the sham Tuina group.The levels of PWTL and PWMT of the Tuina group on the 7 th day after surgery were higher than those of the model group and the sham Tuina group(P<0.01).The levels of NLRP3,ASC,Caspase-1 and IL-1βin the model group were higher than those in the normal group and the sham operation group on the 7 th day after surgery(P<0.01).Compared with those of the model group and the sham Tuina group,the Tuina group significantly down-regulated the expression levels of NLRP3,ASC,Caspase-1 and IL-1β(P<0.05).Conclusion Tuina can alleviate hyperalgesia in SNL model rats,and its mechanism is related to down-regulating the expressions of NLRP3,ASC and Caspase-1 protein in the spinal dorsal horn,thereby inhibiting the activation of NLRP3 inflammasome,hindering the maturation and secretion of IL-1β and blocking the immune inflammatory response which it mediates.