基于星形胶质细胞活化与其介导的P38MAPK/CREB信号通路探讨麦粒灸对坐骨神经损伤大鼠的镇痛机制

Discussion on the relieving pain mechanism of wheat grain moxibustion on sciatic nerve injury rats based on astrocyte activation and its mediated P38MAPK/CREB signaling pathway

目的:观察麦粒灸对坐骨神经损伤(SNI)大鼠腰(L)4-5脊髓中星形胶质细胞(AS)活化、CX43、炎性因子及炎症信号通路的影响,探讨麦粒灸治疗坐骨神经痛的可能机制。方法:SD雄性大鼠随机分为空白组、假手术组、模型组、麦粒灸组,每组6只。模型组、麦粒灸组采用经典钳夹法制备大鼠坐骨神经重度挤压伤模型;假手术组仅暴露坐骨神经,不行钳夹处理;造模后第7天麦粒灸组取患侧环跳穴行麦粒灸治疗,每次灸6壮,以大鼠不全出现全身挣扎为度;其余各组正常喂养,仅抓取固定,不做其他处理;连续干预10d,1次/d。观察造模后第7天、干预结束后的各组大鼠的冷刺激缩足持续时间、纤维丝测痛仪测量各组大鼠的痛阈值;ELISA法检测大鼠脊髓组织中白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)的表达水平,Western Blot法检测大鼠脊髓组织中p38丝裂原活化蛋白酶(P38MAPK)、P-P38、胶质纤维丝酸性蛋白(GFAP)、环磷腺苷效应元件结合蛋白(CREB)、P-CREB、CX43蛋白的表达水平。结果:造模后干预前,与假手术组比较,模型组、麦粒灸组大鼠的机械痛阈值显著降低(P<0.01),冷刺激缩足时间显著升高(P<0.01);干预后,与模型组比较,麦粒灸组大鼠的机械痛阈值显著上升(P<0.01),冷刺激缩足时间显著减短(P<0.01);麦粒灸组大鼠脊髓组织中的IL-1β、IL-6、TNF-α、IFN-γ水平及CX43、GFAP、P-P38、P-CREB蛋白表达均显著降低(P<0.01,P<0.05)。结论:麦粒灸环跳穴可改善SNI大鼠疼痛反应,其机制可能是通过减少CX43、IFN-γ分泌,抑制AS活化,减少促炎因子释放,抑制炎症信号通路激活,发挥镇痛作用。

Objective:To observe the effects of wheat grain moxibustion on astrocyte(AS)activation,gap junction protein 43(CX43),inflammatory factors and inflammatory signalling pathways in the lumbar(L)4-5 spinal cord of rats with sciatic nerve injury(SNI),and to explore the possible mechanism of wheat grain moxibustion in the treatment of sciatica.Methods:Male SD rats were randomly divided into blank group,sham-operated group,model group,and wheatgrain moxibustion group,with 6 rats in each group.The model group and the wheat grain moxibustion group used the classical clamp method to prepare the rat sciatic nerve severe compression injury model;the sham-operated group only exposed the sciatic nerve,not clamp treatment;the wheat grain moxibustion group took the affected side of the Huantiao(GB 30)to carry out the wheat grain moxibustion treatment in the 7th day after the modelling,and moxibustion was performed 6 times each time,so as to ensure that rats did not appear to struggle all over the body;the rest of the groups were fed normally,and were only fixed in the grasp,and did not do any other treatments;the intervention was carried out continuously for 10 d,1 time/d.The duration of cold stimulation and foot reduction in each group of rats on the 7th day after modelling and at the end of the intervention was observed,the pain threshold of rats in each group measured by fibre-wire nociceptor;the expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6),tumour necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)in the spinal cord tissue of rats were detected by ELISA,and the expression levels of interferon-γ(IFN-γ)in the spinal cord tissue of rats were detected by Western Blot.The expression levels of p38 mitogen-activated protease(P38MAPK),P-P38,glial fibrillary acidic protein(GFAP),cyclophosphoadenosine effector binding protein(CREB),P-CREB,and CX43 proteins were detected by Western Blot method in rat spinal cord tissues.Results:Before the intervention after modelling,compared with the sham-operated group,the mechanical pain thresholds of the rats in the model group and the wheat grain moxibustion group decreased(P<0.01),and the time of foot reduction by cold stimulation was elevated(P<0.01).After the intervention,compared with the model group,the rats in the wheat grain moxibustion group showed a significant increase in the thresholds of mechanical pain(P<0.01),and the time of foot reduction by cold stimulation was shortened(P<0.01).The expressiones of IL-1β,IL-6,TNF-α,IFN-γin the spinal cord tissues and the protein expressiones of CX43,GFAP,P-P38 and P-CREB in the spinal cord tissues were decreased(P<0.01,P<0.05).Conclusion:The mechanism of treating Huantiao(GB 30)by wheat grain moxibustion can improve the pain response in SNI rats,which may be through the reduction of CX43,IFN-γsecretion,inhibition of AS activation,reduction of pro-inflammatory factor release,inhibition of inflammatory signalling pathway activation,and play an analgesic role.