脊髓小胶质细胞Toll样受体4和κ阿片受体的相互作用参与针刺镇痛的机制研究

κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats

目的:观察电针对慢性神经病理性疼痛大鼠脊髓小胶质细胞Toll样受体4(TLR4)和κ阿片受体(KOR)表达的影响,探讨脊髓小胶质细胞自身受体的相互作用参与电针镇痛的机制。方法:Wistar大鼠随机分为正常组、模型组、电针组和电针+抑制剂组,每组18只。结扎大鼠坐骨神经造成慢性压迫性损伤(CCI)疼痛模型。电针组、电针+抑制剂组于CCI术后第8天开始电针双侧"足三里""阳陵泉"(1 mA,2 Hz/15 Hz),30 min/次,1次/d,连续5 d;电针+抑制剂组于首次电针前给予大鼠腹腔注射KOR抑制剂JDTic(10 mg/kg)。用足底测痛仪测定大鼠双足对热刺激的缩足反应潜伏期(PWL);用免疫荧光法检测脊髓腰(L)2—L4段中小胶质细胞的形态和数量;用放射免疫法检测脊髓中强啡肽的含量;免疫磁珠法分离脊髓L2—L4段中的小胶质细胞后,用荧光定量PCR法检测脊髓和小胶质细胞中CD68和KOR mRNA的表达;用Western blot法检测小胶质细胞中TLR4蛋白的表达。结果:与正常组比较,模型组大鼠患健侧PWL差值(PWLD)明显增大(P<0.01),脊髓背角小胶质细胞标志物Iba-1和CD68 mRNA表达及脊髓小胶质细胞上TLR4蛋白表达均上升(P<0.01),脊髓背角小胶质细胞胞体增大、突起增多。干预后与模型组比较,电针组大鼠的PWLD减小(P<0.05),脊髓背角Iba-1和CD68mRNA表达降低(P<0.05),小胶质细胞胞体变小,强啡肽含量升高(P<0.05),小胶质细胞上TLR4蛋白表达下降(P<0.05),而KOR mRNA表达升高(P<0.05)。干预后与电针组比较,电针+抑制剂组大鼠的PWLD明显增大(P<0.05),脊髓背角Iba-1、CD68mRNA表达升高(P<0.05),小胶质细胞上TLR4蛋白表达升高(P<0.05),而KOR mRNA表达降低(P<0.05)。结论:电针对慢性神经病理性疼痛的镇痛机制可能与增强脊髓小胶质细胞上KOR mRNA表达,从而抑制小胶质细胞上TLR4诱导的致痛信号有关。

Objective To observe the effect of electroacupuncture(EA)on the expression of lumbar spinalκ-opioid receptor(KOR)and Toll-like receptor 4(TLR4)in microglia in neuropathic pain rats,so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain.Methods Wistar male rats were randomized into control,model,EA and EA plus KOR inhibitor(EA+inhibitor)groups(n=18 in each group).The neuropathic pain model was established in rats by ligature of the right sciatic nerve.EA was applied at bilateral“Zusanli”(ST36)and“Yanglingquan”(GB34)for 30 min,once daily for 5 days.JDTic dihydrochloride(a KOR inhibitor)was administrated by intraperitoneal injection before EA intervention.The difference value of paw withdrawal thermal latency(PWLD)of the bilateral hind-limbs was used as the thermal pain reaction level.At the end of experiments,the rat’s lumbar spinal cord(L2—L4)was taken for detecting the expression of CD68 mRNA(a marker of the activated microglia)and Iba-1(a marker for the activated and resting microglia)immunoactivity,and dynorphin content,and KOR mRNA and TLR4 protein(in immunomagnetic microbead method separated microglia)by using fluorescence quantitative PCR,immunofluorescence,radioimmunoassay and Western blot,separately.Results Compared with the control group,a strong thermal hyperalgesia was induced,the expression levels of Iba-1 and CD68 mRNA in the spinal cord,TLR4 protein of the spinal microglia were significantly increased(P<0.01)in the model group.The microglia were characterized by somatic hypertrophy and thickened branches in the model group.After EA intervention,the PWLD,the expression of Iba-1,CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05),while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05).The hypertrophic microglia shrinked slightly in the EA group.After injection of KOR inhibitor,the PWLD and expression levels of Iba-1,CD68 mRNA and TLR4 protein were significantly increased(P<0.05),and the expression of KOR mRNA was significantly decreased(P<0.05)in the EA+inhibitor group in comparison with the EA group.Conclusion The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.