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Title: [The anti-inflammatory effect of electroacupuncture in mice with spinal cord injury and molecular mechanism based on transcriptome sequencing technology].
Author: Yang ZX, Huang SQ, Tang CL, Zhao HD, Dai P, He XL, Li SJ, Li MJ.
Journal: Zhen Ci Yan Jiu; 2023 Jul 25; 48(7):672-80. PubMed ID: 37518961.
Abstract:
OBJECTIVE: To observe the effect of electroacupuncture(EA) on neural function and spinal cord pathological morphology in spinal cord injury(SCI) mice and investigate the anti-inflammatory molecular mechanism of EA on SCI mice from the aspects of gene by using bioinformatics. METHODS: Seventy-two female C57BL/6 mice were randomized into sham operation, model and EA groups, with 24 mice in each group. The SCI model was established by clamping the spinal cord with a serrefine after laminectomy at the 1^st lumbar vertebra(L1). EA(1.5 Hz/7.5 Hz, 1.0 mA) was applied to bilateral "Jiaji"(EX-B2) and "Zusanli"(ST36) for 10 min, once a day for 14 consecutive days. Basso Mouse Scale(BMS) score was used to assess the hindlimb locomotor function of mice. Histopathological changes of the injured area of the spinal cord were determined by HE staining. The spinal cord RNA was sequenced by using RNA-Seq technology. The bioinformatic analysis was then performed to detect the diffe-rential genes between groups, and the function classification and the involved pathways were enriched. The mRNA and protein expressions of differential genes were detected and verified by using qRT-PCR and Western blot. RESULTS: Compared with the sham operation group, BMS score of the model group was significantly decreased(P<0.05), while that of EA group was increased relevant to the model group (P<0.05). HE staining showed loose and disordered structure and arrangement, cavitation, more inflammatory infiltration, nucleus pycnosis, and neuronal necrosis in the model group, which was alleviated in the EA group. Compared with the sham operation group, 565 differential genes were detected in the model group, including 545 up-regulated and 20 down-regulated, while 41 were detected between the EA and the model group, including 2 up-regulated and 39 down-regulated in the EA group. Fifteen genes that were all up-regulated after modeling and down-regulated after EA intervention were detected by using Venn plot, which are Retn, Adipoq, Myh1, Actn2, Pck1, Klhl41, Fabp4, Hspb7, Myot, Ankrd2, Hrc, Cox6a2, Obscn, Col2a1, Mybpc1, and 3 inflammation-related genes(Fabp4, Adipoq and Pck1) were finally acquired. The 15 differential genes were annotated into main biological processes, cell composition and molecular function in the GO function classification analysis. The 15 differential genes were then enriched into different KEGG pathways, including the peroxisome proliferatorsactivated receptor (PPAR) signaling pathway, Adipocytokine signaling pathway. The mRNA and protein expressions of Fabp4, Adipoq and Pck1 in spinal cord detected by qRT-PCR and Western blot were significantly increased in the model group (P<0.001, P<0.01), while these were significantly decreased in the EA group relevant to the model group(P<0.001, P<0.01, P<0.05). CONCLUSION: EA can promote the repair of nerve function and improve inflammatory infiltration in SCI mice. The mechanism may be closely related to the down-regulation of inflammatory factors Fabp4, Adipoq and Pck1 expression, and the regulation of PPAR and Adipocytokine signaling pathways. 目的：观察电针对脊髓损伤小鼠神经功能及病理形态的影响，从基因和生物信息学层面探讨电针对脊髓损伤小鼠的抗炎作用及相关分子机制。方法：雌性C57BL/6小鼠随机分为假手术组、模型组及电针组,每组24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电针双侧“足三里”“夹脊”,每次10 min,每日1次,连续14 d。采用Basso Mouse Scale(BMS)评分评估小鼠后肢运动功能变化；HE染色法观察脊髓损伤区病理形态；RNA-Seq技术筛选差异基因并进行生物信息学分析；采用荧光定量PCR和Western blot法验证部分差异基因的mRNA及蛋白的表达。结果：与假手术组相比，模型组BMS评分降低(P<0.05)；HE染色显示模型组脊髓损伤区结构疏松紊乱，出现空洞，炎性细胞浸润严重，细胞核固缩，神经元坏死；模型组共筛选出565个差异基因(包括545个上调和20个下调)，其中与炎性免疫相关的基因为脂肪酸结合蛋白4(Fabp4)、脂联素(Adipoq)、磷酸烯醇式丙酮酸羧激酶1(Pck1)，其mRNA表达量均显著升高(P<0.001)，蛋白表达量亦显著升高(P<0.01，P<0.001)。与模型组相比，电针组BMS评分升高(P<0.05)；HE染色显示电针组脊髓结构较完整，组织空洞及炎性浸润减少，正常神经元增多；电针组共筛选出41个差异基因(包括2个上调和39个下调)；Fabp4、Adipoq、Pck1 mRNA表达量显著降低(P<0.001，P<0.01)，蛋白表达量亦显著降低(P<0.05)。Venn分析共获得15个共表达差异基因：Retn、Adipoq、Myh1、Actn2、Pck1、Klhl41、Fabp4、Hspb7、Myot、Ankrd2、Hrc、Cox6a2、Obscn、Col2a1、Mybpc1；GO注释分析显示差异基因主要分为生物学过程、细胞组分、分子功能3类，KEGG通路分析显示差异基因主要富集于过氧化物酶体增殖物激活受体(PPAR)信号通路、脂肪细胞因子(Adipocytokine)信号通路等。结论：电针能促进脊髓损伤小鼠神经功能的修复，改善炎性浸润，其机制可能与下调炎性因子Fabp4、Adipoq、Pck1表达，调控PPAR、Adipocytokine信号通路密切相关。.

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