中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (6): 1152-1158.doi: 10.12307/2025.297

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

补气活血合剂干预脑缺血再灌注模型大鼠相关因子及自噬蛋白的表达

陈玉宁1，蒋  颖1，廖翔宇1，陈琼君1，熊  亮1，刘  悦1，2，刘  通1，2   

  1. 1广州中医药大学第五临床医学院，广东省广州市  510095；2广东省第二中医院针灸康复科，广东省广州市  510095

  • 收稿日期:2023-10-11 接受日期:2024-03-06 出版日期:2025-02-28 发布日期:2024-06-20
  • 通讯作者: 刘通，博士，副主任中医师，博士生导师，广州中医药大学第五临床医学院，广东省广州市 510095；广东省第二中医院针灸康复科，广东省广州市 510095 通讯作者：刘悦，主任中医师，教授，博士生导师，广州中医药大学第五临床医学院，广东省广州市 510095；广东省第二中医院针灸康复科，广东省广州市 510095
  • 作者简介:陈玉宁，女，1998年生，广东省东莞市人，汉族，在读硕士，主要从事脑卒中的临床及机制研究。
  • 基金资助:
    广东省中医药局专项研究项目(20203001)，项目负责人：刘悦；广东省自然科学基金面上项目(2022A1515011676)，项目负责人：刘悦；广东省自然科学基金省企联合基金(2022A1515220012)，项目负责人：刘通；国家自然科学基金面上项目(82174482)，项目负责人：刘通；中央财政转移支付地方项目(602023057)，项目参与人：刘悦，刘通；2024广东省刘悦名中医工作室建设项目(粤中医办函 [2023]108号)，项目负责人：刘悦

Buqi Huoxue Compounds intervene with the expression of related factors and autophagy related proteins in a rat model of cerebral ischemia/reperfusion

Chen Yuning1, Jiang Ying1, Liao Xiangyu1, Chen Qiongjun1, Xiong Liang1, Liu Yue1, 2, Liu Tong1, 2   

  1. 1The Fifth Clinical Medical School of Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China; 2Department of Acupuncture and Rehabilitation, Guangdong Second Hospital of Traditional Chinese Medicine, Guangzhou 510095, Guangdong Province, China 
  • Received:2023-10-11 Accepted:2024-03-06 Online:2025-02-28 Published:2024-06-20
  • Contact: Liu Tong, MD, Associate chief physician, Doctoral supervisor, The Fifth Clinical Medical School of Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China; Department of Acupuncture and Rehabilitation, Guangdong Second Hospital of Traditional Chinese Medicine, Guangzhou 510095, Guangdong Province, China Co-corresponding author: Liu Yue, Chief physician, Professor, Doctoral supervisor, The Fifth Clinical Medical School of Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China; Department of Acupuncture and Rehabilitation, Guangdong Second Hospital of Traditional Chinese Medicine, Guangzhou 510095, Guangdong Province, China
  • About author:Chen Yuning, Master candidate, The Fifth Clinical Medical School of Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China
  • Supported by:
    The Special Research of the Traditional Chinese Medicine Bureau of Guangdong Province, No. 20203001 (to LY); the Natural Science Foundation of Guangdong Province (General Program), No. 2022A1515011676 (to LY); the Natural Science Foundation of Guangdong Province (Provincial-Enterprise Joint Fund), No. 2022A1515220012 (to LT); the National Natural Science Foundation of China, No. 82174482 (to LT); Central Finance Transfer Local Project, No. 602023057 (to LY and LT [project participants]); Guangdong Liu Yue Famous Chinese Medicine Workshop Construction Project, No. [2023]108 (to LY)

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摘要：




文题释义：
补气活血合剂：以益气活血、化痰开窍为治法，由黄芪、赤芍、当归、何首乌、桃仁、瓜蒌子、半夏、陈皮、莱菔子、石菖蒲等药物组成，全方具有补气活血、消痰通络的作用。 
自噬：是细胞修复受损蛋白质和细胞器通路并通过溶酶体降解，从而稳定细胞内环境的生理过程。自噬在许多生理和病理过程中发挥着核心作用，自噬相关蛋白主要有 Beclin-1、LC3B和p62等。Beclin-1触发自噬，LC3B形成自噬体，而 p62与自噬活性呈负相关。

背景：补气活血合剂治疗气虚痰瘀型缺血性脑卒中有显著的临床疗效，然而其具体起效机制尚不明确。
目的：观察补气活血合剂对脑缺血再灌注模型大鼠血管内皮细胞生长因子、碱性成纤维细胞生长因子、脑源性神经营养因子及自噬蛋白Beclin1、p62表达的影响。
方法：取40只雄性SD大鼠，采用随机数字表法分为假手术组、模型组、补气活血合剂组与自噬抑制剂组，每组10只。模型组、补气活血合剂组与自噬抑制剂组建立大脑缺血再灌注损伤模型，补气活血合剂组大鼠再灌注2 h后灌胃给予补气活血合剂(6.49 g/kg，每天分3次给药)，自噬抑制剂组大鼠再灌注2 h后灌胃给予补气活血合剂(6.49 g/kg，每天分3次给药)+腹腔注射自噬抑制剂3-甲基腺嘌呤(灌胃前2 h注射，灌胃第1-3天注射)，假手术组、模型组灌胃给予等量生理盐水，连续灌胃给药7 d。末次给药24 h后，进行大鼠神经功能、脑梗死体积、脑组织形态及大脑缺血皮质区血管内皮细胞生长因子、碱性成纤维细胞生长因子、脑源性神经营养因子及自噬蛋白Beclin1、p62表达检测。
结果与结论：①Zea-Longa评分结果显示，造模后大鼠神经功能受到严重损伤，补气活血合剂组大鼠神经功能损伤轻于模型组、自噬抑制剂组(P < 0.05)；②TTC染色结果显示，模型组、补气活血合剂组与自噬抑制剂组大鼠均可见大脑梗死灶，补气活血合剂组大鼠脑梗死体积低于模型组、自噬抑制剂组(P < 0.05)；③缺血侧脑组织苏木精-伊红染色结果显示，模型组神经细胞间有较大的空隙且排列不整齐，神经细胞出现胞质凝集、核固缩现象；补气活血合剂组和自噬抑制剂组神经细胞间隙减小且排列较规律，存在部分细胞出现胞质凝集、核固缩表现；④免疫组化染色结果显示，血管内皮生长因子多表达在神经元细胞、神经胶质细胞及毛细血管内皮；碱性成纤维细胞生长因子和脑源性神经营养因子多表达在神经元细胞、神经胶质细胞，4组间血管内皮细胞生长因子、碱性成纤维细胞生长因子、脑源性神经营养因子表达比较差异无显著性意义(P > 0.05)；⑤Western blot检测结果显示，与假手术组相比，模型组Beclin1蛋白表达降低(P < 0.05)，p62蛋白表达升高(P < 0.05)；与模型组相比，补气活血合剂组Beclin1蛋白表达升高(P < 0.05)，p62蛋白表达降低(P < 0.05)；与补气活血合剂组相比，自噬抑制剂Beclin1蛋白表达降低(P < 0.05)，p62蛋白表达升高(P < 0.05)；⑥结果表明，补气活血合剂可通过促进自噬来减轻大鼠脑缺血再灌注损伤。
https://orcid.org/0009-0001-2487-6483(陈玉宁)

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: 补气活血合剂, 缺血再灌注, 神经功能缺损, 自噬, 血管内皮生长因子, 碱性成纤维细胞生长因子, 脑源性神经营养因子, Beclin1, p62

Abstract:
BACKGROUND: Buqi Huoxue Compounds have significant clinical efficacy in treating ischemic stroke with Qi deficiency and phlegm stasis; however, the exact mechanism of action is not clear.
OBJECTIVE: To observe the effect of Buqi Huoxue Compounds on the expression of vascular endothelial growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor and autophagy related protein Beclin1 and p62 in a rat model of cerebral ischemia/reperfusion.
METHODS: Forty male Sprague-Dawley rats were randomly divided into sham operation group, model group, Buqi Huoxue Compounds group and autophagy inhibitor group, with 10 rats in each group. In the latter three groups, a rat model of cerebral ischemia/reperfusion injury was established. The Buqi Huoxue Compounds group was intragastrically given Buqi Huoxue Compounds (6.49 g/kg, administered three times a day) 2 hours after reperfusion; the autophagy inhibitor group was intragastrically given Buqi Huoxue Compounds (6.49 g/kg, administered three times a day) 2 hours after reperfusion and intraperitoneally given 3-methyladenine 2 hours before gavage and at days 1-3 of gavage. The sham operation group and model group were given equal amounts of saline by gavage for 7 consecutive days. Neurological function, cerebral infarct volume, brain tissue morphology and expression of vascular endothelial growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor and autophagy-related proteins Beclin1 and p62 in the ischemic cortical region of rats were detected at 24 hours after the final administration.
RESULTS AND CONCLUSION: Zea-Longa scoring results showed that the neurological function of rats was severely damaged after modeling and neurological deficit of rats in the Buqi Huoxue Compounds group was less than that in the model group and the autophagy inhibitor group (P < 0.05). TTC staining showed that cerebral infarct foci were observed in the model group, Buqi Huoxue Compounds group, and autophagy inhibitor group, and the cerebral infarct volume in the Buqi Huoxue Compounds group was lower than that in the model group and the autophagy inhibitor group (P < 0.05). The results of hematoxylin-eosin staining in ischemic brain tissues showed that there were large gaps between nerve cells in the model group and cell arrangement was not neat, and cytoplasmic agglutination and pyknosis were observed. Immunohistochemical staining results showed that vascular endothelial growth factor was mostly expressed in neuronal cells, glial cells and capillary endothelium; basic fibroblast growth factor and brain-derived neurotrophic factor were mostly expressed in neuronal cells and glial cells; and there was no significant difference in the expression of vascular endothelial growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor among the four groups (P > 0.05). The results of western blot assay showed that compared with the sham operation group, Beclin1 protein expression was decreased (P < 0.05) and p62 protein expression was elevated (P < 0.05) in the model group; compared with the model group, Beclin1 protein expression was increased (P < 0.05) and p62 protein expression was reduced (P < 0.05) in the Buqi Huoxue Compounds group; compared with the Buqi Huoxue Compounds group, Beclin1 protein expression was decreased (P < 0.05) and p62 protein expression was elevated (P < 0.05) in the autophagy inhibitor group. To conclude, Buqi Huoxue Compounds attenuate cerebral ischemia-reperfusion injury in rats by promoting autophagy.

Key words: Buqi Huoxue Compounds, ischemia/reperfusion, neurologic deficit, autophagy, vascular endothelial growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor, Beclin1, p62

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