中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (29): 6180-6186.doi: 10.12307/2025.762

• 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神经功能、p-Akt及血清外泌体的表达

刘  通1，2，黄志宾1，陈玉宁1，蒋  颖1，廖翔宇1，陈琼君1，熊  亮1，刘  悦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:2024-06-11 接受日期:2024-09-06 出版日期:2025-10-18 发布日期:2025-03-01
  • 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:第一作者：刘通，博士，副主任中医师，博士生导师，主要从事针灸在脑卒中及脊柱术后康复中的临床及机制研究。 并列第一作者：黄志宾，在读硕士，主要从事针灸推拿学方向的研究。
  • 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；广东省自然基金面上项目 (2022A1515011676)，项目负责人：刘悦；
    广东省自然基金省企联合基金项目 (2022A1515220012)，项目负责人：刘通；国家自然基金面上项目 (82174482)，项目负责人：刘通；中央财政转移支付地方项目(602023057)，项目负责人：刘悦；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 in neurological function, p-Akt and serum exosome expression in a rat model of cerebral ischemia-reperfusion injury

Liu Tong1, 2, Huang Zhibin1, Chen Yuning1, Jiang Ying1, Liao Xiangyu1, Chen Qiongjun1, Xiong Liang1, Liu Yue1, 2   

  1. 1The Fifth Clinical Medical College, 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:2024-06-11 Accepted:2024-09-06 Online:2025-10-18 Published:2025-03-01
  • Contact: Liu Yue, Chief physician, Professor, Doctoral supervisor, The Fifth Clinical Medical College, 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:Liu Tong, PhD, Associate chief physician, Doctoral supervisor, The Fifth Clinical Medical College, 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 Huang Zhibin, Master candidate, The Fifth Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China. Liu Tong and Huang Zhibin contributed equally to this work.
  • 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 LT); 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、制何首乌、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。 
外泌体：具有传递细胞间信息、靶向运输等作用的纳米级囊泡，在脑血管相关疾病的治疗中具有重要作用。

背景：补气活血合剂在治疗气虚痰瘀型缺血性脑卒中方面显示出显著的临床效果，但其具体作用机制有待进一步明确。
目的：探究补气活血合剂对脑缺血再灌注模型大鼠神经功能、p-Akt及血清外泌体表达的影响。
方法：40只6周龄雄性SPF级SD大鼠随机分配至假手术组、模型组、合剂组、合剂+GW4869组，每组10只。模型组、合剂组、合剂+
GW4869组使用线栓法建立大脑中动脉栓塞大鼠模型，假手术组给予切开分离、不插线栓。缺血2 h后拔除线栓进行再灌注，再灌注2 h后，合剂组予补气活血合剂灌胃(6.49 g/kg，分3次给药)，合剂+GW4869组在造模后前3 d，每次灌胃补气活血合剂前2 h给予外泌体抑制剂GW4869腹腔注射[2.5 mg/(kg•d)]，假手术组与模型组灌胃等量生理盐水，3次/d，连续7 d。干预7 d后，检测各组大鼠神经功能、脑梗死体积、脑组织形态、大脑梗死区p-Akt、血清外泌体浓度表征及血清外泌体标志蛋白CD63、TSG101的表达水平。
结果与结论：①造模后，与假手术组相比，模型组、合剂组、合剂+GW4869组神经功能评分均显著升高(P < 0.01或P < 0.05)；干预7 d后，合剂组评分较模型组及合剂+GW4869组显著降低(均P < 0.01)；②2，3，5-氯化三苯基四氮唑染色结果显示，模型组、合剂组以及合剂+GW4869组中皆发现缺血侧梗死灶；与模型组和合剂+GW4869组大鼠脑梗死体积比较，合剂组显著缩小(P < 0.01)；③苏木精-伊红结果表明，合剂组和合剂+GW4869组相较于模型组神经细胞的形态结构异常程度较轻，但仍有部分细胞显示出核固缩和胞质凝集的特征；④纳米颗粒跟踪分析技术分析结果显示，分离出的颗粒直径从 60-300 nm不等，与外泌体的特征大小范围一致；⑤Western blot结果显示，与假手术组相比，模型组梗死区p-Akt及血清外泌体标志蛋白CD63、TSG101表达水平明显降低(P < 0.05或P < 0.01)；与模型组相比，合剂组梗死区p-Akt及血清外泌体标志蛋白CD63、TSG101表达水平明显升高(P < 0.05或P < 0.01)；与合剂组相比，加用GW4869后，梗死区p-Akt及血清外泌体标志蛋白CD63表达水平明显下降(P < 0.05)，TSG101表达有下降趋势，但无明显统计学差异；⑥提示补气活血合剂可能通过促进外泌体释放增强缺血侧皮质区p-Akt的表达，改善脑缺血再灌注模型大鼠神经损伤。 
https://orcid.org/0000-0002-5185-2188(刘通)

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, p-Akt, 外泌体, 工程化组织构建

Abstract: BACKGROUND: Buqi Huoxue Compounds have shown significant clinical effects on the treatment of ischemic stroke due to qi deficiency and phlegm stasis, but its specific mechanism of action needs to be further clarified.
OBJECTIVE: To observe the effects of Buqi Huoxue Compounds on the neurological function, p-Akt and serum exosome expression in a rat model of cerebral ischemia-reperfusion injury. 
METHODS: Forty adult male SPF Sprague-Dawley rats, 6 weeks old, were randomly divided into sham operation group, model group, Buqi Huoxue Compounds group, Buqi Huoxue Compounds+GW4869 group, with 10 rats in each group. In the latter three groups, a rat model of cerebral ischemia-reperfusion injury was established using thread technique. The sham operation group was given incision and separation without inserting a suture. 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 GW4869+Buqi Huoxue Compounds group was intragastrically given Buqi Huoxue Compounds (6.49 g/kg, administered three times a day) 2 hours after reperfusion and intraperitoneally given GW4869 [2.5 mg/(kg•d)] 2 hours before gavage with 3 days after modeling. Both the sham operation group and model group received equal amounts of saline via gavage, three times a day, for 7 consecutive days. Neurological function, cerebral infarct volume, brain tissue morphology, characterization of serum exosome, p-Akt in the cortical area and CD63 and TSG101 in serum exosome were detected after 7 days of administration.
RESULTS AND CONCLUSION: (1) After modeling, compared with the sham operation group, the neurological function scores of the model group, Buqi Huoxue Compounds group, and Buqi Huoxue Compounds+GW4869 group were significantly increased (P < 0.01 or P < 0.05). After 7 days of intervention, the neurological function scores of the Buqi Huoxue Compounds group were significantly lower than those of the model group and Buqi Huoxue Compounds+ GW4869 group (all P < 0.01). (2) The results of 2,3,5-triphenyltetrazolium chloride staining showed that cerebral infarct foci were observed in the model group, Buqi Huoxue Compounds group, and Buqi Huoxue Compounds+GW4869 group, and the cerebral infarct volume in the Buqi Huoxue Compounds group was smaller than that in the model group and the Buqi Huoxue Compounds+GW4869 group (P < 0.01). (3) The results of hematoxylin-eosin staining showed that the morphological and structural abnormalities of nerve cells in the Buqi Huoxue Compounds group and Buqi Huoxue Compounds+GW4869 group were less severe than those in the model group, but some cells still exhibited cytoplasmic agglutination and pyknosis in the Buqi Huoxue Compounds group and Buqi Huoxue Compounds+GW4869 group. (4) NanoSight analysis showed that the diameters of the isolated particles ranged from 60 nm to 300 nm, consistent with the characteristic size of exosomes. (5) Western blot results showed that the expression of p-Akt in the infarct zone and expression of CD63 and TSG101 in serum exosomes were significantly lower in the model group compared with the sham operation group (P < 0.05 or P < 0.01). Compared with the model group, the expression of p-Akt in the infarct zone and expression of CD63 and TSG101 in serum exosomes were significantly higher in the Buqi Huoxue Compounds group than in the model group (P < 0.05 or P < 0.01). However, the p-Akt expression in the infarct zone and CD63 expression in serum exosomes decreased significantly in the Buqi Huoxue Compounds+GW4869 group (P < 0.05), while TSG101 expression decreased without significant difference after the addition of GW4869. (6) To conclude, Buqi Huoxue Compounds attenuate cerebral ischemia-reperfusion injury and increase the expression of p-Akt in rats by promoting exosome secretion.

Key words: Buqi Huoxue Compounds, ischemia-reperfusion, neurological function, p-Akt, exosome, engineered tissue construction

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