中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (22): 3561-3566.doi: 10.12307/2023.360

• 骨科植入物相关基础实验 Basic experiments of orthopedic implant • 上一篇    下一篇

川芎嗪对大鼠脊髓损伤后铁代谢的影响

范  筱1，2，3，陶经纬1，蒋昇源1，邓博文1，穆晓红1   

  1. 1北京中医药大学东直门医院，北京市   100700；2北京中医药大学，北京市   100029；3青岛市市立医院，山东省青岛市   266011
  • 收稿日期:2022-03-31 接受日期:2022-06-23 出版日期:2023-08-08 发布日期:2022-11-02
  • 通讯作者: 穆晓红，博士，主任医师，北京中医药大学东直门医院，北京市 100700
  • 作者简介:范筱，男，1988年生，山东省青岛市人，汉族，2018年福建中医药大学毕业，博士，主治医师，主要从事中医药治疗脊髓损伤的临床及基础研究。
  • 基金资助:
    国家自然科学基金(81874467)，项目负责人：穆晓红；山东省中医药科技发展项目(2019-0614)，项目负责人：范筱；中国博士后基金(2022M710472)，项目负责人：范筱

Effect of tetramethylpyrazine on iron metabolism after spinal cord injury in rats

Fan Xiao1, 2, 3, Tao Jingwei1, Jiang Shengyuan1, Deng Bowen1, Mu Xiaohong1   

  1. 1Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China; 2Beijing University of Chinese Medicine, Beijing 100029, China; 3Qingdao Municipal Hospital, Qingdao 266011, Shandong Province, China
  • Received:2022-03-31 Accepted:2022-06-23 Online:2023-08-08 Published:2022-11-02
  • Contact: Mu Xiaohong, MD, Chief physician, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China
  • About author:Fan Xiao, MD, Attending physician, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China; Beijing University of Chinese Medicine, Beijing 100029, China; Qingdao Municipal Hospital, Qingdao 266011, Shandong Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 81874467 (to MXH); Science and Technology Development Project of Traditional Chinese Medicine of Shandong Province, No. 2019-0614 (to FX); China Postdoctoral Fund, No. 2022M710472 (to FX)

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摘要：

文题释义：

铁死亡：是一种铁依赖性的，区别于细胞凋亡、细胞坏死、细胞自噬的新型细胞程序性死亡方式。铁死亡的主要机制是在二价铁或酯氧合酶的作用下，催化细胞膜上高表达的不饱和脂肪酸，发生脂质过氧化，从而诱导细胞死亡；此外，还表现为抗氧化体系(谷胱甘肽系统)的调控核心酶GPX4的降低。
脊髓损伤：由创伤暴力作用于脊柱，引起脊柱骨折、脱位，从而导致脊髓和马尾神经受损，损伤平面以下肢体出现运动功能、感觉功能、神经反射以及括约肌功能障碍的严重中枢神经系统损伤。

背景：铁代谢紊乱是导致脊髓损伤后神经细胞铁死亡的重要病理因素，不利于脊髓损伤修复。川芎嗪作为行气活血中药川芎的有效活性成分单体，被证实对脊髓损伤具有良好的抗炎、抗脂质过氧化反应以及神经保护作用，需进一步明确其促进脊髓损伤修复的作用机制。
目的：研究川芎嗪对大鼠脊髓损伤后铁代谢的调节作用，探讨其改善脊髓损伤的相关机制。
方法：将36只SD大鼠随机分为假手术组、模型组和川芎嗪组，每组12只；假手术组仅行椎板切除术，术后给予生理盐水腹腔注射；模型组和川芎嗪组制备脊髓损伤模型，术后分别给予生理盐水和川芎嗪腹腔注射，术后4周取材。采用BBB肢体运动功能评分评价大鼠肢体运动功能，尼氏染色观察神经元形态，普鲁士染色观察脊髓组织铁沉积，铁检测试剂盒检测脊髓组织铁含量，免疫组化检测铁蛋白重链1和铁蛋白轻链表达，Western blot检测铁蛋白重链1和铁蛋白轻链的蛋白表达量。
结果与结论：①模型组和川芎嗪组大鼠各个时间点BBB评分显著低于假手术组(P < 0.01)，自术后第14天，川芎嗪组大鼠BBB评分显著高于模型组(P < 0.01)；②尼氏染色结果显示，假手术组神经元形态结构正常，模型组可见大量瘀血，神经元形态结构紊乱，川芎嗪组瘀血较少，神经元形态结构较模型组改善；③普鲁士染色结果显示，假手术组铁沉积较少，模型组和川芎嗪组铁沉积较多；普鲁士染色阳性平均吸光度值模型组和川芎嗪组显著大于假手术组(P < 0.01)，川芎嗪组显著小于模型组(P < 0.01)；④铁含量检测结果显示，模型组和川芎嗪组显著多于假手术组(P < 0.01)，川芎嗪组显著少于模型组(P < 0.01)；⑤免疫组化染色结果显示，铁蛋白重链1和铁蛋白轻链阳性表达平均吸光度值模型组和川芎嗪组显著小于假手术组(P < 0.01)，川芎嗪组显著大于模型组(P < 0.01)；⑥Western blot检测结果显示，铁蛋白重链1蛋白和铁蛋白轻链蛋白的相对表达量模型组和川芎嗪组显著少于假手术组(P < 0.01)，川芎嗪组显著多于模型组(P < 0.01)；⑦结果说明，川芎嗪通过调控铁蛋白重链1和铁蛋白轻链的表达而调节脊髓损伤后铁代谢紊乱，从而发挥神经保护作用，促进脊髓损伤大鼠肢体运动功能恢复。

https://orcid.org/0000-0001-5147-6701 (范筱) 

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

关键词: 脊髓损伤, 川芎嗪, 铁代谢, 铁死亡, 铁蛋白重链1, 铁蛋白轻链

Abstract: BACKGROUND: Iron metabolism disorder is an important pathological factor leading to ferroptosis of nerve cells after spinal cord injury, which is not conducive to the repair of spinal cord injury. Tetramethylpyrazine, as the active ingredient monomer of Ligusticum Chuanxiong, a traditional Chinese medicine for activating qi and activating blood, has been proven to have good anti-inflammatory, anti-lipid peroxidation and neuroprotective effects on spinal cord injury. It is necessary to further clarify its mechanism of promoting spinal cord injury repair.
OBJECTIVE: To study the regulation mechanism of tetramethylpyrazine on iron metabolism after spinal cord injury in rats, and to explore its related mechanism of improving spinal cord injury.
METHODS: Totally 36 Sprague-Dawley rats were randomly divided into sham operation group, model group and tetramethylpyrazine group, with 12 rats in each group. In the sham operation group, only laminectomy was performed and normal saline was injected intraperitoneally after operation. Spinal cord injury models were prepared in model group and tetramethylpyrazine group. Normal saline and tetramethylpyrazine were injected intraperitoneally after operation, and the tissues were taken 4 weeks after operation. BBB limb motor function score was used to evaluate the limb motor function of rats. Nissl staining was used to observe the morphology of neurons. Prussian staining was used to observe the iron deposition in spinal cord tissue. Iron detection kit was used to detect the iron content in spinal cord tissue. Immunohistochemistry was used to detect the expression of ferritin heavy chain 1 and ferritin light chain protein. Western blot assay was used to detect the expression of ferritin heavy chain 1 and ferritin light chain protein.  
RESULTS AND CONCLUSION: (1) The BBB score of model group and tetramethylpyrazine group was significantly lower than that of sham operation group at each time point (P < 0.01). Since the 14th day after operation, the BBB score of tetramethylpyrazine group was significantly higher than that of model group (P < 0.01). (2) Nissl staining results showed that the morphology and structure of neurons in the sham operation group were normal; a large amount of blood stasis and disordered morphology and structure of neurons were observed in the model group; blood stasis was less and morphology and structure of neurons were improved in the tetramethylpyrazine group. (3) Prussian staining results showed that there was less iron deposition in the sham operation group, and more iron deposition in the model group and tetramethylpyrazine group. The average optical density of positive Prussian staining in the model group and tetramethylpyrazine group was significantly higher than that in the sham operation group (P < 0.01). The average optical density of positive Prussian staining in tetramethylpyrazine group was significantly lower than that in the model group (P < 0.01). (4) The detection of iron content showed that the iron content in model group and tetramethylpyrazine group was significantly higher than that in sham operation group (P < 0.01). The iron content in tetramethylpyrazine group was significantly lower than that in model group (P < 0.01). (5) Immunohistochemical results showed that the average optical density of positive ferritin heavy chain 1 and ferritin light chain protein expression in model group and tetramethylpyrazine group was significantly lower than that in sham operation group (P < 0.01). The average optical density of positive ferritin heavy chain 1 and ferritin light chain protein expression in tetramethylpyrazine group was significantly higher than that in model group (P < 0.01). (6) Western blot assay showed that the relative expression of ferritin heavy chain 1 protein and ferritin light chain protein in model group and tetramethylpyrazine group was significantly lower than that in sham operation group (P < 0.01). The relative expression of ferritin heavy chain 1 protein and ferritin light chain protein in tetramethylpyrazine group was significantly higher than that in model group (P < 0.01). (7) It is concluded that tetramethylpyrazine can regulate the disorder of iron metabolism after spinal cord injury by regulating the expression of ferritin heavy chain 1 and ferritin light chain protein, so as to play a neuroprotective role and promote the recovery of limb motor function in spinal cord injury rats.

Key words: spinal cord injury, tetramethylpyrazine, iron metabolism, ferroptosis, ferritin heavy chain 1, ferritin light chain protein

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