针刺研究

2021, v.46(09) 735-741

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脊髓Toll样受体4和热休克蛋白90参与电针缓解大鼠慢性神经病理性疼痛机制研究
Spinal cord Toll like receptor 4 and its co-stimulatory molecule heat shock protein 90 may participate in electroacupuncture analgesia in rats with chronic neuropathic pain

高永辉;王俊英;韩焱晶;刘俊岭;
GAO Yong-hui;WANG Jun-ying;HAN Yan-jing;LIU Jun-ling;Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences;

摘要(Abstract):

目的:观察电针对坐骨神经慢性压迫性损伤(CCI)大鼠脊髓中Toll样受体4(TLR4)及热休克蛋白90(HSP90)表达的影响,探讨脊髓TLR4及HSP90参与电针缓解慢性神经病理性疼痛的机制。方法:Wistar大鼠随机分为正常组、模型组、电针组、抑制剂组和电针+抑制剂组。结扎大鼠坐骨神经造成CCI疼痛模型。电针组于CCI术后7 d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;抑制剂组以及电针+抑制剂组分别于每日及每日电针前给予大鼠颈部皮下注射HSP90抑制剂格尔德霉素(50μg/kg),连续5 d。用足底测痛仪测定大鼠双足缩腿反应潜伏期(PWL);用Western blot法检测腰(L)2—L4段脊髓中TLR4和HSP90蛋白的表达;酶联免疫吸附法检测大鼠L2—L4段脊髓中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的含量;用免疫荧光双标记法检测大鼠L2—L4段脊髓中TLR4在小胶质细胞和神经元上的分布。结果:干预后,与正常组比较,模型组大鼠患健侧PWL差值(PWLD),脊髓中TLR4和HSP90表达,IL-1β和TNF-α含量,离子钙接头蛋白分子1(Iba1)和TLR4共表达细胞比例明显增大(P<0.01,P<0.05)。与模型组比较,电针组、抑制剂组和电针+抑制剂组大鼠的PWLD,IL-1β和TNF-α含量,Iba1和TLR4共表达细胞比例明显降低(P<0.05);电针组HSP90的表达明显升高(P<0.05),神经元核抗原(NeuN)和TLR4共表达细胞比例明显升高(P<0.05);抑制剂组和电针+抑制剂组TLR4和HSP90表达明显降低(P<0.05)。与电针组比较,抑制剂和电针+抑制剂组大鼠的PWLD,TLR4和HSP90表达,NeuN和TLR4以及Iba1和TLR4共表达细胞比例明显降低(P<0.05)。电针+抑制剂组NeuN和TLR4共表达细胞比例高于抑制剂组(P<0.05)。结论:电针"足三里""阳陵泉"穴可缓解CCI大鼠的神经病理性疼痛,其效应可能与对脊髓小胶质细胞和神经元中TLR4的调节有关,脊髓中HSP90可能是电针通过调节TLR4抑制神经病理性疼痛的协同刺激分子。
Objective To observe the effect of electroacupuncture(EA) on the expression of Toll like receptor 4(TLR4)and heat shock protein 90(HSP90) in the spinal cord of rats with chronic constriction injury(CCI) of sciatic nerve, so as to explore the mechanism of spinal cord TLR4 and HSP90 in alleviating chronic neuropathic pain by EA. Methods Male Wistar rats were randomized into control, model, EA, HSP90 inhibitor(inhibitor) and EA+ inhibitor groups(n=10 in each group). The neuropathic pain model was established by ligature of the right sciatic nerve to induce CCI. EA(1 mA,2 Hz/15 Hz)was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. Rats of the inhibitor and EA+inhibitor groups were given a subcutaneous injection of HSP90 inhibitor geldanamycin(50 μg/kg) at the neck before daily EA. The paw withdrawal latency(PWL) of the bilateral hind-limbs was detected by using an algesia-detector. The contents of interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the lumbar spinal cord(L2—L4) tissue were detected by enzyme-linked immunosorbent assay. The relative expression levels of HSP90 and TLR4 proteins in the lumbar spinal cord(L2—L4) were detected using Western blot and immunofluorescence double labeling, respectively. Results Following CCI, a strong thermal hyperalgesia, an apparent up-regulation of expression of HSP90 and TLR4 proteins and TLR4 in microglia, and increasing levels of IL-1β and TNF-α in the spinal cord were induced in the model group relevant to the control group(P<0.01,P<0.05). Five sessions of EA intervention or inhibitor injection significantly attenuated hyperalgesia, reversed the increase of IL-1β and TNF-α, and down-regulated the expression of TLR4 in microglia(P<0.05). Compared with the model group, the expression of HSP90 was further increased(P<0.05), and those of TLR4 in microglia and neurons were significantly decreased and increased, respectively in the EA group(P<0.05). Compared with the EA group, the levels of PWLD,TLR4 and HSP90 expression, and the proportions of neuronal nuclei antigen(NeuN) and TLR4, and ionized calcium binding adapter molecule(Iba1) and TLR4 co-expressed cells were significantly decreased in the inhibitor group and EA+inhibitor group(P<0.05). The proportion of NeuN and TLR4 co-expression cells in the EA+inhibitor group was significantly higher than that of the inhibitor group(P<0.05). Conclusion EA stimulation of ST36 and GB34 can alleviate thermal hyperalgesia in CCI rats, which is closely associated with its effect in regulating the expression of TLR4 in the spinal cord neurons and microglia. HSP90 in the spinal cord may be a co-stimulatory molecule for EA induced relief of neuropathic pain by regulating TLR4.

关键词(KeyWords): 慢性神经病理性疼痛;针刺镇痛;Toll样受体4;热休克蛋白90;小胶质细胞;神经元
Chronic neuropathic pain;Acupuncture analgesia;Toll-like receptor 4;Heat shock protein 90;Microglia;Neuron

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(No.81473779);; 国家重点基础研究发展计划“973”中医专项(No.2013CB531904)

作者(Author): 高永辉;王俊英;韩焱晶;刘俊岭;
GAO Yong-hui;WANG Jun-ying;HAN Yan-jing;LIU Jun-ling;Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences;

Email:

DOI: 10.13702/j.1000-0607.201103

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