中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (26): 4139-4146.doi: 10.12307/2023.596

• 神经组织构建 nerve tissue construction • 上一篇    下一篇

“通督调神”电针预处理大鼠缺血半暗带区miR-124和皮质区蛋白的表达

张君宇1，张利达2，胡滟琦3，金子开4，罗佛赐5，吴晓晴6，童婷婷6，宋小鸽7，韩  为1   

  1. 1安徽中医药大学第二附属医院，安徽省合肥市  230001；2 广州医科大学附属脑科医院中医科，广东省广州市  510370；3 安徽中医药大学针灸推拿学院，安徽省合肥市  230031；4 中国中医科学院望京医院，北京市  100102；5 江苏省第二中医院，江苏省南京市  210017；6 安徽中医药大学研究生院，安徽省合肥市  230022；7 安徽省针灸经络研究所，安徽省合肥市  230031
  • 收稿日期:2022-06-28 接受日期:2022-10-12 出版日期:2023-09-18 发布日期:2023-01-28
  • 通讯作者: 韩为，博士，主任中医师，安徽中医药大学第二附属医院，安徽省合肥市 230001
  • 作者简介:张君宇，男，1991年生，安徽省阜阳市人，汉族，2018年安徽中医药大学毕业，硕士，主治医师，主要从事通督调神针刺治疗脑血管疾病方面的研究。 张利达，男，1994年生，安徽省舒城县人，汉族，2021年安徽中医药大学毕业，硕士，医师，主要从事针药结合防治脑病的临床和机制研究。

Expression of microRNA-124 and cortical proteins in ischemic penumbra of rats pretreated with “Tongdu Tiaoshen” electroacupuncture

Zhang Junyu1, Zhang Lida2, Hu Yanqi3, Jin Zikai4, Luo Fuci5, Wu Xiaoqing6, Tong Tingting6, Song Xiaoge7, Han Wei1   

  1. 1The Second Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230001, Anhui Province, China; 2Department of Traditional Chinese Medicine, Affiliated Brain Hospital of Guangzhou Medical University, Guangzhou 510370, Guangdong Province, China; 3College of Acupuncture, Moxibustion and Tuina, Anhui University of Traditional Chinese Medicine, Hefei 230031, Anhui Province, China; 4Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100102, China; 5The Second Hospital of Traditional Chinese Medicine of Jiangsu Province, Nanjing 210017, Jiangsu Province, China; 6Graduate School of Anhui University of Traditional Chinese Medicine, Hefei 230022, Anhui Province, China; 7Anhui Acupuncture and Meridian Research Institute, Hefei 230031, Anhui Province, China
  • Received:2022-06-28 Accepted:2022-10-12 Online:2023-09-18 Published:2023-01-28
  • Contact: Han Wei, MD, Chief physician, The Second Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230001, Anhui Province, China
  • About author:Zhang Junyu, Master, Attending physician, The Second Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230001, Anhui Province, China Zhang Lida, Master, Physician, Department of Traditional Chinese Medicine, Affiliated Brain Hospital of Guangzhou Medical University, Guangzhou 510370, Guangdong Province, China

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摘要：

文题释义：

“通督调神”针法：为张道宗教授多年临证经验的凝练总结，是指通过针刺督脉穴位为主，辅以心包经穴位的特殊治疗方法。以“通督、调神、康复”为原则，主要作用于脑-心-肾-督轴，在激发督脉经气运行的同时，能发挥局部取穴的效果，直接作用于脑，从而达到通调经络、调和营卫、平衡阴阳的作用。
脑缺血再灌注损伤：脑组织缺血后，缺血区血液的再灌注早期有助于减轻脑组织损伤、恢复神经功能，随后会产生氧化应激、钙离子超载、神经元兴奋性氨基酸毒性增强、炎性反应等过程，导致继发性损伤，即脑缺血再灌注损伤，该损伤可诱发更加严重的神经细胞凋亡和脑功能障碍。

背景：“通督调神”电针预处理法是结合传统针刺和现代电刺激的新兴物理治疗方法，既往文献报道其对缺血性脑卒中具有显著治疗作用，但其具体机制尚不明确。
目的：探讨“通督调神”电针预处理作用于细胞凋亡，对大鼠缺血半暗带区miR-124和皮质区Notch-1、p-JNK和半胱氨酸天冬氨酸特异性蛋白酶3蛋白相对表达量的影响及其可能的作用机制。
方法：将75只雄性SD大鼠随机分为模型组、假手术组、电针预处理组、miR-124抑制剂预处理组和电针+miR-124激动剂预处理组，每组15只。7 d 干预期内，电针预处理组取“风府”“百会”“大椎”穴行电针，1次/d，20 min/次；miR-124抑制剂预处理组第7天注射miR-124 antagomir至大鼠侧脑室内；电针+miR-124激动剂预处理组第7天在电针基础上注射miR-124 agomir；模型组及假手术组不予治疗。干预结束后造模，假手术组只做鼠板上固定和钝性分离血管，其余组均采用大脑中动脉闭塞方法并参照Zea Longa线栓法建立大鼠右侧脑缺血再灌注损伤模型。造模完成后24 h应用改良神经损伤程度评分定损，评分结束后使用TUNEL染色法测细胞凋亡，电镜观察大鼠皮质脑区超微结构，Western Blot法检测皮质区蛋白相对表达量，实时荧光定量PCR检测mRNA相对表达量。

结果与结论：①与假手术组比较，其余各组大鼠改良神经损伤程度评分均升高(均P < 0.01)；与模型组比较，miR-124抑制剂预处理组和电针预处理组改良神经损伤程度评分显著降低(均P < 0.05)；②与假手术组比较，模型组大鼠凋亡细胞数量显著升高(P < 0.01)；与模型组比较，各预处理组大鼠凋亡细胞数量显著降低(均P < 0.01)；③模型组细胞结构及内容物缺损较重；电针预处理组细胞结构及内容物轻度缺损；miR-124抑制剂预处理组细胞较完整；电针+miR-124激动剂预处理组细胞结构及内容物存在损伤；④与模型组比较，miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区半胱氨酸天冬氨酸特异性蛋白酶3、Notch-1和p-JNK蛋白相对表达量显著降低(均P < 0.01)；⑤与模型组比较，各预处理组大鼠皮质脑区miR-124-3p和Notch-1 mRNA相对表达量降低(均P < 0.01)，miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区半胱氨酸天冬氨酸特异性蛋白酶3 mRNA和p-JNK mRNA相对表达量降低(均P < 0.01)，电针+miR-124激动剂预处理组大鼠皮质脑区p-JNK mRNA相对表达量也降低(P < 0.05)；⑥结果表明，“通督调神”电针预处理可通过干预脑缺血再灌注损伤大鼠脑组织内miR-124表达水平，抑制脑缺血再灌注损伤后神经元细胞凋亡，发挥减轻脑缺血再灌注损伤神经功能缺损的作用，其作用机制可能与miR-124对Notch通路的正向调节机制有关。

https://orcid.org/0000-0001-9003-9889(张君宇)

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: 脑缺血再灌注, 细胞凋亡, microRNA-124, 通督调神, Notch通路

Abstract: BACKGROUND: “Tongdu Tiaoshen” electroacupuncture pretreatment is an emerging physiotherapy combining traditional acupuncture with modern electrical stimulation, which has been reported to have significant therapeutic effects on ischemic stroke, but its specific mechanism is still unclear.
OBJECTIVE: To investigate the effects of “Tongdu Tiaoshen” electroacupuncture pretreatment on cell apoptosis, the relative expression of microRNA-124 (miR-124) in the ischemic penumbra and Notch-1, p-JNK and cysteinyl-aspartate specific protease-3 (caspase-3) proteins in the cortex of rats and their possible mechanisms of action.
METHODS: Seventy-five male Sprague-Dawley rats were randomly divided into five groups (n=15 per group): model group, sham-operated group, electroacupuncture pretreatment group, miR-124 antagomir pretreatment group and electroacupuncture+miR-124 agomir pretreatment group. In the electroacupuncture pretreatment group, 20-minute electroacupuncture at Fengchi, Baihui, and Dazhui was performed once a day, for 7 continuous days. In the miR-124 antagomir pretreatment group, miR-124 antagomir was injected into the lateral ventricle of rats on day 7. In the electroacupuncture+miR-124 agomir pretreatment group, miR-124 agomir was injected based on electroacupuncture pretreatment. Model and sham-operated groups were not treated. After the intervention, animal models were created. In the sham-operated group, the rats were only fixed on the operating board and the vessels were blunt-separated. In the other groups, the right cerebral ischemia-reperfusion injury model was established by the middle cerebral artery occlusion method and the Zea Longa suture method. Twenty-four hours after the modeling, the modified neurological severity scoring was performed to determine the damage. TUNEL staining method was then used to measure cell apoptosis in the rat cerebral cortex. Ultrastructure of the rat cerebral cortex was observed under electron microscopy.
RESULTS AND CONCLUSION: Compared with the sham-operated group, the modified neurological severity scores were significantly elevated in the other groups (all P < 0.01). Compared with the model group, the miR-124 antagomir pretreatment group and electroacupuncture pretreatment group had significantly reduced modified neurological severity scores (both P < 0.05). Compared with the sham-operated group, the number of apoptotic cells was elevated in the model group (P < 0.01). Compared with the model group, the number of apoptotic cells was reduced in the pretreatment groups (all P < 0.01). The model group had more severe defects in cell structure and contents. The electroacupuncture pretreatment group had mild defects in cell structure and contents. Cells in the miR-124 antagomir pretreatment group were more intact. There was damage to cell structure and contents in the electroacupuncture+miR-124 agomir pretreatment group. The relative expressions of caspase-3, Notch-1 and p-JNK proteins in the cortex of rats were significantly lower in the miR-124 antagomir pretreatment and electroacupuncture pretreatment group compared with the model group (all P < 0.01). The relative expression of miR-124-3p and Notch-1 mRNA in the cortex of the pretreated rats was significantly reduced compared with that of the model group (all P < 0.01). The relative expression of caspase-3 mRNA and p-JNK mRNA in the cortex of rats was significantly reduced in the miR-124 antagomir pretreatment and electroacupuncture pretreatment groups compared with the model group (all P < 0.01). The relative expression of p-JNK mRNA in the cortex of rats in the electroacupuncture+miR-124 agomir pretreatment group was also reduced compared with the model group (P < 0.05). To conclude, “Tongdu Tiaoshen” electroacupuncture pretreatment can inhibit the apoptosis of neuronal cells after cerebral ischemia-reperfusion injury by interfering with the expression level of miR-124 in the brain tissue of rats, thereby reducing neurological deficits after cerebral ischemia-reperfusion injury. The mechanism of this action may be related to the positive regulation mechanism of miR-124 on the Notch pathway.

Key words: cerebral ischemia-reperfusion injury, apoptosis, microRNA-124, Tongdu Tiaoshen, Notch signaling pathway

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