中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (6): 1118-1126.doi: 10.12307/2025.302

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

温肾通督方促进小鼠脊髓损伤的修复

赵瑞华1，2，陈思娴1，2，郭  杨2，3，石  磊1，2，吴承杰2，吴  毛3，杨光露2，张昊恒2，马  勇1，2，3   

  1. 1南京中医药大学中医学院•中西医结合学院，江苏省南京市  210023；2南京中医药大学骨伤修复与重建新技术实验室，江苏省南京市  210023；3南京中医药大学无锡附属医院，江苏省中医退行性骨关节病临床医学创新中心，江苏省无锡市  214071
    第一作者：赵瑞华，女，2000年生，山东省潍坊市人，汉族，南京中医药大学在读硕士，主要从事脊髓损伤修复方面的研究工作。
    通讯作者：马勇，教授，博士生导师，南京中医药大学中医学院•中西医结合学院，江苏省南京市  210023；南京中医药大学骨伤修复与重建新技术实验室，江苏省南京市  210023；南京中医药大学无锡附属医院，江苏省中医退行性骨关节病临床医学创新中心，江苏省无锡市  214071
  • 收稿日期:2024-01-10 接受日期:2024-03-13 出版日期:2025-02-28 发布日期:2024-06-20
  • 通讯作者: 马勇，教授，博士生导师，南京中医药大学中医学院•中西医结合学院，江苏省南京市 210023；南京中医药大学骨伤修复与重建新技术实验室，江苏省南京市 210023；南京中医药大学无锡附属医院，江苏省中医退行性骨关节病临床医学创新中心，江苏省无锡市 214071
  • 作者简介:赵瑞华，女，2000年生，山东省潍坊市人，汉族，南京中医药大学在读硕士，主要从事脊髓损伤修复方面的研究工作。
  • 基金资助:
    国家自然科学基金项目(81973885)，项目负责人：马勇；国家自然科学基金项目(82174400)，项目负责人：吴毛；江苏省中医退行性骨关节病临床医学创新中心资助项目(苏中医科教[2021]4号)，项目负责人：马勇；国家中医药管理局高水平中医药重点学科建设项目资助(国中医药人教函[2023]85号)，项目负责人：马勇

Wen-Shen-Tong-Du Decoction promoting spinal cord injury repair in mice 

Zhao Ruihua1, 2, Chen Sixian1, 2, Guo Yang2, 3, Shi Lei1, 2, Wu Chengjie2, Wu Mao3, Yang Guanglu2, Zhang Haoheng2, Ma Yong1, 2, 3   

  1. 1School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; 2Laboratory of New Techniques of Restoration & Reconstruction, Institute of Traumatology & Orthopedics, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; 3Jiangsu CM Clinical Innovation Center of Degenerative Bone & Joint Disease, Wuxi TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Wuxi 214071, Jiangsu Province, China 
  • Received:2024-01-10 Accepted:2024-03-13 Online:2025-02-28 Published:2024-06-20
  • Contact: Ma Yong, Professor, Doctoral supervisor, School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; Laboratory of New Techniques of Restoration & Reconstruction, Institute of Traumatology & Orthopedics, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; Jiangsu CM Clinical Innovation Center of Degenerative Bone & Joint Disease, Wuxi TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Wuxi 214071, Jiangsu Province, China
  • About author:Zhao Ruihua, Master candidate, School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; Laboratory of New Techniques of Restoration & Reconstruction, Institute of Traumatology & Orthopedics, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • Supported by:
    National Natural Science Foundation of China, Nos. 81973885 (to MY) and 82174400 (to WM); a grant from Jiangsu Provincial Chinese Medicine Innovation Center for Clinical Degenerative Osteoarticular Disease, No. [2021]4 (to MY); High-level Chinese Medicine Key Disciplines Construction Project of National Administration of Traditional Chinese Medicine, No. [2023]85 (to MY)

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摘要：




文题释义：
PI3K/Akt信号通路：具有调节细胞增殖和凋亡的作用，其中PI3K作为肌醇与磷脂酰肌醇的重要激酶，调节突触可塑性和长时程增强，可被外界的多种刺激激活，影响Akt等下级蛋白和细胞的活动过程；Akt被称为丝氨酸/苏氨酸激酶，调控凋亡蛋白抑制凋亡，其活性对神经元发育和神经胶质细胞活动至关重要。
髓系细胞触发受体2：是中枢神经系统中仅在小胶质细胞上表达的蛋白，具有抑制中枢炎症、加强吞噬系统对凋亡细胞碎片的吞噬功能等，其表达与PI3K/Akt信号通路中的关键蛋白有密切关系。

背景：前期研究证实，温肾通督方可通过抑制脾脏B细胞焦亡、促进微血管内皮细胞吞噬髓鞘碎片、影响小胶质细胞迁移和浸润、促进受损神经元恢复、降低脊髓损伤后神经元凋亡等促进脊髓损伤恢复，但其机制尚不明确。
目的：探讨温肾通督方对脊髓损伤小鼠髓系细胞触发受体2(triggering receptor expressed on myeloid cells 2，TREM2)及PI3K/Akt信号通路的影响。
方法：取36只C57BL/6 小鼠，采用随机数字表法分为假手术组、模型组、温肾通督方组，每组12只。模型组与温肾通督方组采用改良Allen’s法制备小鼠T10脊髓损伤模型，造模后第1天，温肾通督方组灌胃给予温肾通督方，假手术组、模型组灌胃给予生理盐水，每天1次，连续给药28 d。给药期间，通过BMS评分和斜板实验评价各组小鼠运动功能；造模后第7，28天，采用苏木精-伊红染色观察各组小鼠脊髓组织病理变化，免疫荧光染色双标法检测脊髓组织离子化钙结合适配分子1(ionized calcium binding adaptor molecule 1，IBA1)、TREM2蛋白表达，Western blot检测脊髓组织TREM2、PI3K、p-PI3K、Akt、p-Akt、Bcl2、Bax、Caspase3蛋白表达。
结果与结论：①BMS评分和斜板实验结果表明，脊髓损伤造模后小鼠后肢运动功能下降，而经过温肾通督方治疗后，脊髓损伤小鼠后肢运动功能明显提升。②苏木精-伊红染色结果显示，与模型组比较，温肾通督方小鼠脊髓组织病理结构明显改善，表现为背侧白质和神经元萎缩程度、细胞质空泡化降低及炎性细胞浸润减少等。③免疫荧光染色结果显示，造模后第7天，模型组IBA1、TREM2蛋白表达均低于假手术组(P < 0.05)，温肾通督方组IBA1、TREM2蛋白表达均高于模型组(P < 0.05)；造模后第28天，模型组TREM2蛋白表达低于假手术组(P < 0.05)，温肾通督方组小鼠脊髓组织中的TREM2蛋白表达高于模型组(P < 0.05)。④Western blot检测结果表明，造模后第7天，与假手术组相比，模型组TREM2、Akt蛋白表达及Bcl2/Bax比值降低(P < 0.05)，p-Akt、Bax蛋白表达及p-Akt/Akt比值升高(P < 0.05)；与模型组相比，温肾通督方组TREM2、PI3K、p-PI3K、Akt、p-Akt、Bcl2蛋白表达及p-PI3K/PI3K、p-Akt/Ak、Bcl2/Bax比值升高(P < 0.05)，Bax、Caspase3蛋白表达降低(P < 0.05)。造模后第28天，与假手术组相比，模型组TREM2、PI3K、p-PI3K、Akt、p-Akt、Bcl2蛋白表达及Bcl2/Bax比值降低(P < 0.05)，Bax蛋白表达升高(P < 0.05)；与模型组相比，温肾通督方组TREM2、PI3K、Akt、p-Akt、Bcl2蛋白表达及Bcl2/Bax比值升高(P < 0.05)，Bax蛋白表达降低(P < 0.05)。⑤结果表明，温肾通督方可能通过上调小胶质细胞TREM2激活PI3K/Akt信号通路，抑制神经元凋亡发挥神经保护作用，进而促进脊髓损伤的修复。
https://orcid.org/0009-0008-2783-0617(赵瑞华)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 脊髓损伤, 温肾通督方, 小胶质细胞, 髓系细胞触发受体2, PI3K/Akt信号通路

Abstract:
BACKGROUND: Previous studies have confirmed that Wen-Shen-Tong-Du Decoction can promote the recovery of spinal cord injury by inhibiting pyroptosis of splenic B cells, promoting the phagocytosis of myelin debris by microvascular endothelial cells, affecting the migration and infiltration of microglia, promoting the recovery of damaged neurons, and decreasing neuronal apoptosis after spinal cord injury, but the mechanism of this is still not clear.
OBJECTIVE: To investigate the effect of Wen-Shen-Tong-Du Decoction on the triggering receptor expressed on myeloid cells 2 (TREM2) and PI3K/Akt signaling pathways in mice following spinal cord injury. 
METHODS: Thirty-six C57BL/6 mice were selected and randomly divided into a sham-operation group, a model group and a Wen-Shen-Tong-Du Decoction group, with 12 mice in each group. In the model and Wen-Shen-Tong-Du Decoction groups, mouse models of T10 spinal cord injury were prepared by the modified Allen’s method. On the 1st day after modeling, the Wen-Shen-Tong-Du Decoction group was given Wen-Shen-Tong-Du Decoction by gavage, and the sham-operation group and the model group were given saline by gavage once a day for 28 days. During the drug administration period, mouse motor function was evaluated by Basso Mouse Scale score and inclined plane test. On the 7th and 28th days after modeling, hematoxylin-eosin staining was used to observe the histopathological changes in the spinal cord tissue of the mice; immunofluorescence double staining was used to detect the protein expression of ionized calcium binding adaptor molecule 1 (IBA1) and TREM2; and western blot assay was used to detect the expression of TREM2, PI3K, p-PI3K, Akt, p-Akt, Bcl2, Bax and Caspase3 in spinal cord tissue. 
RESULTS AND CONCLUSION: Basso Mouse Scale scores and inclined plane test results indicated that the motor function of the mouse hindlimbs was declined after spinal cord injury, and Wen-Shen-Tong-Du Decoction significantly improved motor function in mice with spinal cord injury. Hematoxylin-eosin staining results revealed that Wen-Shen-Tong-Du Decoction significantly ameliorated the pathological structure of spinal cord tissue compared with the model group, manifesting as reduced degrees of dorsal white matter and neuronal atrophy, decreased cytoplasmic vacuolization, and reduced inflammatory cell infiltration. Immunofluorescence double staining results showed that on the 7th day after modeling, the protein expression of IBA1 and TREM2 in the model group was lower than that in the sham-operation group (P < 0.05), and the protein expression of IBA1 and TREM2 in the Wen-Shen-Tong-Du Decoction group was higher than that in the model group (P < 0.05); on the 28th day after modeling, the protein expression of TREM2 in the model group was lower than that in the sham-operation group (P < 0.05), and the protein expression of TREM2 in the spinal cord tissue of the mice in the Wen-Shen-Tong-Du Decoction group was higher than that in the model group (P < 0.05). Western blot results analysis demonstrated that on the 7th day after modeling, compared with the sham-operation group, the model group exhibited a significant reduction in TREM2, PI3K, and Bcl2/Bax (P < 0.05), as well as a significant increase in p-Akt, Bax and p-Akt/Aktp-PI3K (P < 0.05); compared with the model group, the Wen-Shen-Tong-Du Decoction group showed a significant increase in TREM2, PI3K, p-PI3K, Akt, p-Akt, Bcl2, p-PI3K/PI3K, p-Akt/Ak, and Bcl2/Bax (P < 0.05), as well as a significant decrease in Bax and Caspase3 protein expression (P < 0.05). On the 28th day after modeling, compared with the sham-operation group, the model group exhibited a significant reduction in TREM2, PI3K, p-PI3K, Akt, p-Akt, Bcl2 and Bcl2/Bax 
(P < 0.05), as well as a significant increase in Bax protein expression (P < 0.05); compared with the model group, the Wen-Shen-Tong-Du Decoction group showed a significant increase in TREM2, PI3K, Akt, p-Akt, Bcl2, and Bcl2/Bax (P < 0.05), as well as a significant decrease in Bax protein expression (P < 0.05). To conclude, Wen-Shen-Tong-Du Decoction may activate the PI3K/Akt signaling pathway by up-regulating the expression of TREM2 protein in microglia, and then inhibit neuronal apoptosis, thus exerting neuroprotective effects and promoting the repair of spinal cord injury.


Key words: spinal cord injury, Wen-Shen-Tong-Du Decoction, microglia, triggering receptor expressed on myeloid cells 2, PI3K/Akt signaling pathway

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