中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (33): 5327-5333.doi: 10.12307/2023.724

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

山楂叶总黄酮调控星形胶质细胞治疗脊髓损伤的分子机制

马瑞鑫1，刘  槃2，张  琼3，曾高峰3，宗少晖1，2   

  1. 广西医科大学，1再生医学与医用生物资源开发应用省部共建协同创新中心，3公共卫生学院，广西壮族自治区南宁市   530021；2 广西医科大学第一附属医院脊柱骨病外科，广西壮族自治区南宁市   530021
  • 收稿日期:2022-09-08 接受日期:2022-11-18 出版日期:2023-11-28 发布日期:2023-03-30
  • 通讯作者: 宗少晖，教授，主任医师，广西医科大学再生医学与医用生物资源开发应用省部共建协同创新中心，广西壮族自治区南宁市 530021； 广西医科大学第一附属医院脊柱骨病外科，广西壮族自治区南宁市 530021 曾高峰，教授，博士生导师，广西医科大学公共卫生学院，广西壮族自治区南宁市 530021
  • 作者简介:马瑞鑫，男，1995年生，山东省济宁市人，在读硕士，主要从事脊柱脊髓疾病治疗方面的研究。
  • 基金资助:
    国家自然科学基金项目(81860391)，项目负责人：曾高峰；广西医学高层次骨干人才培养“139”计划(桂卫科教发〔2020〕15 号)，项目负责人：宗少晖；广西十百千人才工程资助项目(桂人社发〔2019〕32 号)，项目负责人：曾高峰

Molecular mechanism of total flavonoids of hawthorn leaves regulating astrocytes to repair spinal cord injury

Ma Ruixin1, Liu Pan2, Zhang Qiong3, Zeng Gaofeng3, Zong Shaohui1, 2   

  1. 1Collaborative Innovation Center of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; 2Department of Spinal Osteopathy, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; 3School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Received:2022-09-08 Accepted:2022-11-18 Online:2023-11-28 Published:2023-03-30
  • Contact: Zong Shaohui, Professor, Chief physician, Collaborative Innovation Center of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; Department of Spinal Osteopathy, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China Zeng Gaofeng, Professor, Doctoral supervisor, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • About author:Ma Ruixin, Master candidate, Collaborative Innovation Center of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Supported by:
    National Natural Science Foundation of China, No. 81860391 (to ZGF); The “139” Plan for Training High-Level Backbone Medical Talents in Guangxi Zhuang Autonomous Region, No. [2020]15 (to ZSH); Guangxi Ten Hundred Thousand Talents Project, No. GRSF[2019]32 (to ZGF)

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摘要：


文题释义：

山楂叶总黄酮：是从蔷薇科植物山里红的干燥叶中提取的天然黄酮类化合物，在抗炎症、抗氧化损伤、防止神经退行性病变、预防神经系统疾病和防止中风等方面有较好的效果。
脊髓损伤：是由于各种不同致病因素引起脊髓结构和功能的损伤，可导致严重的运动、感觉和自主神经功能障碍，其主要原因是神经元的损失。在治疗过程中，神经元的修复至关重要，避免因神经元损失后不可再生导致永久性功能丧失。

背景：星形胶质细胞对脊髓损伤后神经元的修复具有重要作用，有研究证明山楂叶总黄酮可促进大鼠脊髓损伤后运动功能恢复，山楂叶总黄酮是否影响星形胶质细胞促进神经元修复仍不清楚。
目的：探索山楂叶总黄酮调控星形胶质细胞治疗脊髓损伤的分子机制。
方法：构建脊髓星形胶质细胞与脊髓神经元 transwell共培养模型，对照组为正常脊髓星形胶质细胞和正常脊髓神经元；损伤组为正常脊髓星形胶质细胞和损伤脊髓神经元；药物组为125 μg/mL山楂叶总黄酮干预的正常脊髓星形胶质细胞和损伤脊髓神经元。荧光探针DCFH-DA检测脊髓神经元内活性氧的生成；实时荧光定量PCR检测脑源性神经营养因子、神经生长因子、转录因子4的表达；Western blot检测脑源性神经营养因子、神经生长因子、Wnt/β-catenin通路关键蛋白GSK-3β、phsopho-GSK-3β(ser9)、β-catenin及转录因子4的表达；免疫荧光观察Wnt/β-catenin通路关键蛋白β-catenin的表达。

结果与结论：①损伤组活性氧生成远高于对照组(P < 0.001)；药物组活性氧生成低于损伤组(P < 0.01)，高于对照组(P < 0.001)；②药物组脑源性神经营养因子、神经生长因子基因及蛋白表达高于对照组与损伤组(P < 0.01)；③与对照组、损伤组相比，药物组phsopho-GSK-3β(ser9)蛋白表达增加(P < 0.001)，β-catenin蛋白表达增加(P < 0.001)，转录因子4基因及蛋白表达增加(P < 0.01)；④结果表明，山楂叶总黄酮可以减轻脊髓神经元的氧化损伤，激活星形胶质细胞Wnt/β-catenin通路，结合转录因子4启动转录，促进其分泌神经营养因子，实现对脊髓神经元的修复。

https://orcid.org/0000-0003-2162-0391 (马瑞鑫) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 山楂叶总黄酮, 星形胶质细胞, 脊髓损伤, Wnt/β-catenin通路, 脑源性神经营养因子, 神经生长因子

Abstract: BACKGROUND: Astrocytes play an important role in neuronal repair after spinal cord injury. Studies have shown that total flavonoids of hawthorn leaves can promote motor function recovery after spinal cord injury in rats. Whether total flavonoids of hawthorn leaves can affect astrocytes to promote neuronal repair remains unclear.  
OBJECTIVE: To explore the molecular mechanism of total flavonoids of hawthorn leaves regulating astrocytes to repair spinal cord injury.
METHODS: Transwell was used to construct the co-culture model of injured spinal cord neurons and spinal cord astrocytes. The control group consisted of normal spinal astrocytes and normal spinal neurons. The injured group consisted of normal astrocytes and injured spinal neurons. The drug group consisted of 125 μg/mL total flavonoids of hawthorn leaves-intervened normal spinal astrocytes and injured spinal neurons. The generation of reactive oxygen species in spinal cord neurons was detected by the fluorescence probe DCFH-DA. Real-time fluorescent quantitative PCR was used to detect the expression of brain-derived neurotrophic factor, nerve growth factor and transcription factor-4. The expression levels of brain-derived neurotrophic factor, nerve growth factor, Wnt/β-catenin pathway key proteins GSK-3β, phsopho-GSK-3β (ser9), β-catenin and transcription factor-4 were detected by western blot assay. The expression of key protein β-catenin of the Wnt/β-catenin pathway was observed by immunofluorescence method.  
RESULTS AND CONCLUSION: (1) The production of reactive oxygen species in the injury group was much higher than that in the control group (P < 0.001). The production of reactive oxygen species in the drug group was lower than that in the injury group (P < 0.01) and higher than that in the control group (P < 0.001). (2) The gene expression of brain-derived neurotrophic factor and nerve growth factor in the drug group was higher than that in the control group and injury group (P < 0.01). (3) Compared with the control group and the injury group, the protein expression of phsopho-GSK-3β(ser9) increased (P < 0.001); β-catenin protein expression increased (P < 0.001); the protein and gene expression of transcription factor 4 was increased (P < 0.01) in the drug group. (4) The results showed that the total flavonoids of hawthorn leaves could alleviate the oxidative damage of spinal cord neurons, activate the Wnt/β-catenin pathway of astrocytes, combine with transcription factor 4 to start transcription, promote its secretion of neurotrophic factors, and realize the repair of spinal cord neurons.

Key words: total flavonoids of hawthorn leaves, astrocyte, spinal cord injury, Wnt/β-catenin pathway, brain-derived neurotrophic factor, nerve growth factor

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