冯韬, 吕烨华, 王盛, 黄维, 糜大国. 黄芪注射液对大鼠急性脊髓损伤的神经保护作用及机制研究[J]. 南京中医药大学学报, 2022, 38(12): 1128-1136. DOI: 10.14148/j.issn.1672-0482.2022.1128
引用本文: 冯韬, 吕烨华, 王盛, 黄维, 糜大国. 黄芪注射液对大鼠急性脊髓损伤的神经保护作用及机制研究[J]. 南京中医药大学学报, 2022, 38(12): 1128-1136. DOI: 10.14148/j.issn.1672-0482.2022.1128
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FENG Tao, LYU Yehua, WANG Sheng, HUANG Wei, MI Daguo. Neuroprotective Effect and Mechanism of Astragalus Injection on Acute Spinal Cord Injury in Rats[J]. Journal of Nanjing University of traditional Chinese Medicine, 2022, 38(12): 1128-1136. DOI: 10.14148/j.issn.1672-0482.2022.1128
Citation: FENG Tao, LYU Yehua, WANG Sheng, HUANG Wei, MI Daguo. Neuroprotective Effect and Mechanism of Astragalus Injection on Acute Spinal Cord Injury in Rats[J]. Journal of Nanjing University of traditional Chinese Medicine, 2022, 38(12): 1128-1136. DOI: 10.14148/j.issn.1672-0482.2022.1128
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黄芪注射液对大鼠急性脊髓损伤的神经保护作用及机制研究

Neuroprotective Effect and Mechanism of Astragalus Injection on Acute Spinal Cord Injury in Rats

    摘要
  • 摘要:
      目的  基于细胞凋亡Fas和TRAIL死亡受体信号通路探讨黄芪注射液对大鼠急性脊髓损伤(SCI)的神经保护作用及机制。
      方法  40只健康的雄性SD大鼠随机分为假手术组，SCI模型组, 黄芪注射液低、中、高剂量(1、2、4 mL · kg-1)组, 每组8只。采用改良的重物打击法构建大鼠急性SCI模型; 按照BBB评分和Rivlin斜板实验评价大鼠运动功能; 苏木精-伊红(Hematoxylin-eosin, HE)染色检测脊髓组织病理变化; 尼氏染色法检测脊髓神经元细胞损伤程度; 高通量转录组测序分析差异表达基因; qPCR验证基因转录水平的表达量; 利用TUNEL试剂盒检测各组脊髓细胞凋亡情况; Western blot检测凋亡通路关键蛋白表达水平。
      结果  BBB评分和斜板试验结果显示, 与假手术组比较, 模型组大鼠表现出明显的运动功能障碍; HE染色显示模型组脊髓组织病理病变严重; 模型组尼氏染色着色较浅, 神经元细胞损伤严重; 与假手术组比较, 模型组脊髓中4 597个基因差异表达, Fas、TRAIL、Caspase-3、Caspase-8、Caspase-9和Bax蛋白表达显著上调(P < 0.01), Fas凋亡抑制分子FAIM和抗凋亡因子Bcl-2的蛋白表达量显著降低(P < 0.01)。与模型组比较, 黄芪注射液组的BBB评分、Rivlin斜板度数显著升高(P < 0.05, P < 0.01), 组织病变程度较小, 尼氏染色着色程度随剂量升高而变深, 黄芪注射液高剂量组脊髓神经细胞损伤和凋亡得到显著缓解(P < 0.01)。转录组测序和qPCR结果显示: 与模型组比较, 黄芪注射液中、高剂量组中参与细胞凋亡调控的FAIM和TRAIL mRNA差异表达(P < 0.01), 黄芪注射液高剂量组Fas、TRAIL、Caspase-3、Caspase-8、Caspase-9和Bax蛋白表达被剂量依赖性地抑制(P < 0.05，P < 0.01), FAIM和Bcl-2的蛋白表达上调(P < 0.01)。
      结论  黄芪注射液可能通过对死亡受体途径Fas和TRAIL蛋白的抑制, 进而调控神经元细胞的程序性凋亡, 发挥对SCI的神经保护作用, 改善脊髓组织损伤病变程度, 加速后期神经运动功能障碍的恢复。

     

    Abstract:
      OBJECTIVE   To explore the neuroprotective function of Astragalus injection on acute spinal cord injury (SCI) in rats and its mechanism based on apoptosis signaling pathway.
      METHODS   40 healthy male SD rats were randomly divided into sham operation group, SCI group, Astragalus injection low dose group (1 mL · kg-1), Astragalus injection medium dose group (2 mL · kg-1) and Astragalus injection high dose group (4 mL · kg-1). A modified heavy object method was used to construct an acute SCI rat model. The locomotor function of rats was evaluated according to BBB method and Rivlin inclined board test. Hematoxylin-eosin (HE) staining was used to detect the pathological changes of spinal cord tissue. Nissl staining method was used to detect the degree of spinal cord neuronal cell damage. High-throughput transcriptome sequencing to analyze differentially expressed genes. The qPCR method verified the expression level of gene transcription. TUNEL kit was used to detect the apoptosis of spinal cord cells in each group. The expression levels of key proteins in the apoptosis pathway was detected by Western blot.
      RESULTS   The results of BBB score and Rivlin plate test indicated that rats in the model group showed obvious motor dysfunction compared with the sham group. HE staining showed severe pathological changes in spinal cord tissue in the model group. Lighter Nissl staining showed severe neuronal cell damage in the model group. Compared with the sham group, 4 597 genes were differentially expressed in the spinal cord of the model group. The expressions of Fas, TRAIL, Caspase-3, Caspase-8, Caspase-9 and Bax proteins were significantly up-regulated (P < 0.01). Moreover, the protein expressions of Fas apoptosis inhibitor FAIM and anti-apoptotic factor Bcl-2 was significantly reduced (P < 0.01). Compared with the model group, the BBB score and the degree of Rivlin in the Astragalus injection group significantly increased and the degree of tissue lesions becomes smaller (P < 0.05, P < 0.01). As the dose of Astragalus injection increased, the degree of Nissl staining becomes deeper. The injury and apoptosis of spinal cord nerve cells in the Astragalus injection high dose group significantly alleviated (P < 0.01). Compared with the model group, the expressions of FAIM and TRAIL mRNA were significantly expressed in the high-dose Astragalus injection group (P < 0.01), the expressions of Fas, TRAIL, Caspase-3, Caspase-8, Caspase-9 and Bax protein in Astragalus injection groups were inhibited in a dose-dependent manner (P < 0.05, P < 0.01), the protein expressions of FAIM and Bcl-2 were up-regulated (P < 0.01).
      CONCLUSION   Astragalus injection may inhibit the expressions of Fas and TRAIL proteins, thereby regulate the programmed apoptosis of neuronal cells, exert a neuroprotective effect on SCI, improve the degree of SCI, and accelerate the later neuromotor function recovery of obstacles.

     

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