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Title: [Influence of electroacupuncture on locomotor function and expression of spinal HMGB1 and TLR4 in mice with spinal cord injury].
Author: Zhao HD, Huang SQ, Tang CL, Dai N, Dai P, Tan YL.
Journal: Zhen Ci Yan Jiu; 2021 Apr 25; 46(4):259-65. PubMed ID: 33931988.
Abstract:
OBJECTIVE: To observe the effect of electroacupuncture(EA)on locomotor activity and the expression of high-mobility group box-1(HMGB1) and Toll-like receptor 4(TLR4) in mice with spinal cord injury(SCI), so as to explore its mechanisms underlying improvement of SCI at the acute stage. METHODS: Forty-eight female C57BL/6 mice were equally randomized into 3 groups: sham operation, model and EA. The SCI model was established by clamping the spinal cord with a serrefine after laminectomy at the 1st lumbar vertebra(L1). EA (1.5 Hz/7.5 Hz, 1.0 mA) was applied to "Jiaji"(EXH-B2) for 10 min, once a day for 5 and 14 days, separately. The hindlimb locomotor function was assessed by Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB). Histopathological changes of the injured area of the spinal cord were determined by H.E. staining. The expression levels of spinal HMGB1, TLR4, ionized calcium binding adaptor molecule 1(Iba1) proteins were detected by Western blot, and the Iba1-positive microglial cells and HMGB1 and Iba1 co-labelled microglia were displayed by immunofluorescence staining. RESULTS: After SCI, the BBB scores on day 5 and 14 were obviously decreased (P<0.05), and the expression of HMGB1 on day 14, TLR4 on day 5 and 14, the number of Iba1-positive microglia as well as the co-expressed HMGB1/Iba1-positive microglia on day 5 and 14 were significantly increased in the model group relevant to the sham operation group (P<0.05, P<0.01). In the EA intervention group, SCI-induced reduction of BBB scores on day 5 and 14, and increases of the expression of HMGB1 and Iba1 on day 14, and TLR4 on day 5 and 14, and the number of Iba1-positive cells as well as the co-expressed HMGB1/Iba1-positive microglia on day 14 were reversed relevant to the model group (P<0.05,P<0.01). H.E. staining showed a structural disorder with lots of cavities, severe inflammatory infiltration with a large quantity of inflammatory cells, and a reduction of normal neurons in the injured spinal cord tissue in the model group， which was relatively milder, with lower activation of microglia in the EA group. CONCLUSION: EA can significantly improve locomotor function in SCI mice, which is associated with its effects in suppressing the expression of inflammatory factors such as HMGB1, TLR4, Iba1 and the over-activation of microglia. 目的：观察电针对脊髓损伤小鼠急性期运动功能及高迁移率族蛋白B1(HMGB1)、Toll样受体4(TLR4)等因子表达水平的影响，探讨电针在小鼠运动功能修复中的抗炎机制。方法：将C57BL/6小鼠随机分为假手术组、模型组及电针组，每组16只，再分为5 d和14 d两个亚组，每个亚组8只。采用钳夹法制备脊髓损伤小鼠模型。电针组于造模后3 h电针“夹脊”穴，每次10 min，每日1次，分别治疗5、14 d。采用BBB评分评估小鼠后肢功能运动变化；HE染色法观察小鼠脊髓损伤部位形态学变化；Western blot法检测小鼠脊髓组织HMGB1、TLR4及小胶质细胞特异性抗体钙离子接头蛋白-1(Iba1)的表达水平；免疫荧光染色法检测HMGB1和Iba1共同标记的小胶质细胞数量及小胶质细胞的形态变化。结果：与假手术组比较，造模后模型组BBB评分降低(P<0.05)；HE染色显示脊髓结构紊乱、炎性浸润严重，正常神经元大量减少；造模后第5天脊髓中TLR4蛋白表达水平升高(P<0.05)，造模后第14天脊髓中HMGB1、TLR4蛋白表达水平均升高（P<0.05,P<0.01）；造模后第5、14天脊髓中Iba1阳性细胞数增多（P<0.05）；造模后第5、14天脊髓中HMGB1/Iba1共阳性表达细胞数量增多(P<0.05)；小胶质细胞呈过度激活态。与模型组比较，电针组造模后第5、14天BBB评分显著升高(P<0.05)；脊髓结构紊乱及炎性浸润有所改善；造模后第5、14天脊髓中TLR4蛋白表达水平及造模后第14天脊髓中HMGB1、Iba1蛋白表达水平均显著下降(P<0.05，P<0.01)；造模后第14天脊髓中Iba1阳性细胞数减少（P<0.05）；造模后第14天HMGB1/Iba1共阳性表达细胞数量显著减少(P<0.05)；小胶质细胞形态由过度激活态明显转变为静息态。结论：电针能有效促进脊髓损伤小鼠急性期运动功能的修复，其机制可能与下调炎性因子HMGB1、TLR4及Iba1的表达水平，抑制小胶质细胞过度激活有关。.

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