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    山楂叶总黄酮抑制小胶质细胞活化对脊髓损伤的影响

    Effect of total flavonoids of hawthorn leaves on spinal cord injury via inhibiting microglia activation

      摘要
    • 摘要: 目的：探究山楂叶总黄酮（TFHL）对脂多糖（LPS）活化后的BV2小胶质细胞和脊髓损伤（SCI）大鼠的作用。方法：LPS诱导BV2小胶质细胞活化建立神经炎症模型；实验分为正常对照组、LPS组及TFHL低、中、高剂量组。根据Allen's法建立SCI模型；将大鼠随机分为假手术组、模型组和TFHL组。采用实时荧光定量PCR（RT-qPCR）检测TNF-α、IL-6、IL-1β、iNOS、COX-2的mRNA 表达；流式细胞术检测活性氧（ROS）水平；蛋白免疫印迹检测TNF-α、IL-6 的蛋白表达；Basso-Beattie-Bresnahan（BBB）评分评估SCI大鼠的运动功能；苏木精—伊红（HE）染色观察脊髓组织结构完整性。结果：在细胞实验中，与正常对照组相比，LPS组和TFHL各剂量组TNF-α、IL-6、IL-1β、iNOS、COX-2的mRNA相对表达水平均增高（P< 0.000 1）；TFHL低剂量组的TNF-α、IL-6，TFHL中剂量组TNF-α、IL-1β、IL-6以及TFHL高剂量组的TNF-α、IL-1β、IL-6、iNOS、COX-2的mRNA表达水平均较LPS组降低（P< 0.05）。与正常对照组相比，LPS组和TFHL各剂量组TNF-α、IL-6的蛋白表达量增高（P< 0.000 1），而TFHL中剂量组的IL-6及TFHL高剂量组的TNF-α、IL-6蛋白表达量均较LPS组降低（P< 0.01）。与正常对照组相比，LPS组和TFHL各剂量组ROS水平均升高（P< 0.000 1），而TFHL各剂量组的ROS水平均较LPS组下降（P< 0.000 1）。在动物实验中，与假手术组相比，模型组和TFHL 组BBB 评分均降低（P< 0.000 1），TFHL 组第5 天和第7 天BBB 评分均较模型组增高（P< 0.05）。与模型组相比，TFHL组脊髓病理结构较完整，空洞面积较少。结论：TFHL可通过抑制小胶质细胞的激活，改善炎症微环境，从而发挥保护受损脊髓神经运动功能的作用。

       

      Abstract: Objective:To exolore the effect of total flavonoids of hawthorn leaves(TFHL)on BV2 microglia activated with lipopolysaccharide (LPS) and spinal cord injury (SCI) rats.Methods:BV2 microglia were activated with LPS to establish a neuroinflammatory model.The experiments were divided into normal control group, LPS group and low, medium and high TFHL dose groups.The SCI model was established according to Allen's method.The rats were randomly divided into sham operation group, model group and TFHL group.The mRNA expression levels of TNF-α, IL-1β, IL-6, iNOS, COX-2 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR).The level of reactive oxygen species (ROS) was detected by flow cytometry.Western blotting was used to detect the protein expressions of TNF-α and IL-6.Basso-Beattie-Bresnahan(BBB)score was used to evaluate the motor function of rats with SCI.HE staining was used to observe the structural integrity of the spinal cord.Results:In cell experiments, compared with normal control group, the mRNA expression levels of TNF-α, IL-6, IL-1β, iNOS, COX-2 in LPS group and TFHL dose groups increased (P< 0.0001); the mRNA expression levels of TNF-α and IL-6 in low TFHL dose group, TNF-α, IL-1β and IL-6 in medium TFHL dose group and TNF-α, IL-1β, IL-6, iNOS and COX-2 in high TFHL dose group were lower than those in LPS group(P< 0.05).Compared with normal control group, the protein expression levels of TNF-α and IL-6 in LPS group and TFHL dose groups increased(P< 0.0001), while the protein expression levels of IL-6 in medium TFHL dose group and TNF-α and IL-6 in high TFHL dose group were lower than those in LPS group(P< 0.01).Compared with normal control group, the levels of ROS in LPS group and TFHL dose groups increased(P< 0.0001), while the levels of ROS in TFHL dose groups were lower than those of LPS group(P< 0.0001).In animal experiments, compared with sham operation group, the BBB scores in model group and TFHL group decreased (P< 0.0001), and the BBB scores in TFHL group on the fifth day and the seventh day were higher than those in model group(P< 0.05).Compared with model group, the spinal cord structure of TFHL group was more complete and cavity area was less.Conclusion:TFHL can improve the inflammatory microenvironment by inhibiting the activation of microglia, thus playing a role in protecting the neuromotor function of injured spinal cord.

       

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