通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs的保护作用

戴纪恒; 吴思鹏; 汪宁; 王艳; 吴倩

doi:10.13422/j.cnki.syfjx.20201405

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通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs的保护作用

经典名方 | 更新时间：2020-09-09

- 通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs的保护作用
- Protective Effect of Cerebrospinal Fluid Containing Tongqiao Huoxuetang on Brain Microvascular Endothelial Cells from Rats with Oxygen Glucose Deprivation/Reoxygenation Injury
- 中国实验方剂学杂志 2020年26卷第14期页码：42-52
- 作者机构：
  
  1. 安徽中医药大学药学院，中药复方安徽省重点实验室，合肥 230012
  2. 安徽省中医药科学院中药药效与安全性评价研究所，新安医学教育部重点实验室，合肥 230012
- 作者简介：
  
  戴纪恒，硕士，从事中药药理学研究，E-mail：1215587216@qq.com
  汪宁，博士，教授，博士生导师，从事中药药效物质基础及药理学研究，Tel：0551-68129186，E-mail：wnsci123@163.com
- 基金信息：
  
  国家自然科学基金项目(81773933)
- DOI：10.13422/j.cnki.syfjx.20201405
  中图分类号： R2-0;R22;R285.5;R289
- 纸质出版日期：2020-07-20，
  
  网络出版日期：2020-04-28，
- 稿件说明：
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戴纪恒,吴思鹏,汪宁等.通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs的保护作用[J].中国实验方剂学杂志,2020,26(14):42-52.

DAI Ji-heng,WU Si-peng,WANG Ning,et al.Protective Effect of Cerebrospinal Fluid Containing Tongqiao Huoxuetang on Brain Microvascular Endothelial Cells from Rats with Oxygen Glucose Deprivation/Reoxygenation Injury[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(14):42-52.
戴纪恒,吴思鹏,汪宁等.通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs的保护作用[J].中国实验方剂学杂志,2020,26(14):42-52. DOI： 10.13422/j.cnki.syfjx.20201405.

DAI Ji-heng,WU Si-peng,WANG Ning,et al.Protective Effect of Cerebrospinal Fluid Containing Tongqiao Huoxuetang on Brain Microvascular Endothelial Cells from Rats with Oxygen Glucose Deprivation/Reoxygenation Injury[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(14):42-52. DOI： 10.13422/j.cnki.syfjx.20201405.

摘要

目的

探讨通窍活血汤含药脑脊液对氧糖剥夺/复糖复氧（OGD/R）损伤大鼠脑微血管内皮细胞（BMECs）的保护作用及潜在机制。

方法

通过酶消化法提取原代BMECs，并将细胞随机分为6组，分别为正常组，OGD/R组，通窍活血汤（TQHXT）组（20%），尼莫地平（NMDP）组（10 μmol·L

-1

），卡博替尼（BMS）组（1 μmol·L

-1

）和合用药组。除正常组外，其余各组细胞在氧糖剥夺2 h后迅速复糖复氧24 h进行OGD/R造模并分组给药。采用细胞免疫荧光染色法鉴定BMECs，观察OGD/R损伤大鼠BMECs的形态学和超微结构改变并检测细胞跨膜电阻（TEER）值变化。用试剂盒检测细胞内一氧化氮（NO）水平、乳酸脱氢酶（LDH）活性、活性氧（ROS）荧光强度和组织型纤溶酶原激活因子（tPA）含量。采用流式细胞术检测细胞内钙离子浓度及细胞凋亡，观察血管新生标记因子CD34的表达，用蛋白免疫印迹法（Western blot）检测细胞中紧密连接蛋白（ZO-1），血管内皮生长因子（VEGF），黏着斑激酶（FAK）和桩蛋白（Paxillin）蛋白的表达情况。

结果

与正常组比较，OGD/R组细胞皱缩、变圆，细胞TEER值和细胞中ZO-1蛋白表达显著降低，细胞中NO，LDH及ROS水平显著升高，tPA含量显著降低，细胞中钙离子浓度和细胞凋亡显著增加，细胞中CD34有所表达，VEGF，FAK和Paxillin蛋白表达显著升高（

0.01）；与OGD/R组比较，TQHXT组细胞损伤明显改善，细胞TEER值和细胞中ZO-1蛋白表达显著升高，细胞中NO，LDH及ROS含量显著降低，tPA含量显著升高，细胞中钙离子浓度和细胞凋亡显著减少，细胞中CD34表达增加，VEGF，FAK和Paxillin蛋白表达显著升高（

0.05，

0.01）。

结论

通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs具有保护作用，该保护作用可能是通过VEGF/VEGF受体2（R

）/FAK/Paxillin信号通路促进血管生成来发挥作用。

Abstract

Objective

To investigate the protective effect of cerebrospinal fluid containing Tongqiao Huoxuetang (TQHXT) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (BMECs)

in order to explore the underlying mechanisms.

Method

Primary BMECs were extracted by enzymatic digestion

and the cells were randomly divided into six groups: the normal control group

the OGD/R group

the TQHXT group（20%）

the nimodipine（NMDP） group (10 μmol·L

-1

)

the cabozanix group (1 μmol·L

-1

) and the combination group. Except for the normal control group

the cells in the other groups were rapidly reoxygenated for 24 h after 2 h of oxygen-glucose deprivation

the OGD/R modeling was performed

and the rats were administered with drugs by groups. BMECs were identified by cell immunofluorescence staining

morphological and ultrastructural changes of OGD/R-induced BMECs were observed

and changes in cell transmembrane resistance (TEER) were detected. The levels of nitric oxide (NO)

the activity of lactate dehydrogenase (LDH)

the fluorescence intensity of reactive oxygen species (ROS) and the content of tissue-type plasminogen activator (tPA) were measured with kits. Intracellular Ca

concentration and cell apoptosis were detected by flow cytometry

and the expression of CD34 was observed. The protein expressions of zonula occluden-1 (ZO-1)

vascular endothelial growth factor (VEGF)

adhesion kinase (FAK)

and Paxillin were detected by Western blot.

Result

Compared with the normal control group

the cells in the OGD/R group were shrinking and rounded

TEER value and ZO-1 protein expression in cells were significantly decreased

the contents of NO

LDH and ROS in cells were significantly increased

the content of tPA was significantly decreased

the concentration of Ca

and the apoptosis in the cells were significantly increased

CD34 was expressed in cells

and the protein expressions of VEGF

FAK and Paxillin were significantly increased (

0.01). Compared with the OGD/R group

cell damage in the TQHXT group was significantly improved

the TEER value and ZO-1 protein expression in cells were significantly increased

the contents of NO

LDH and ROS in cells were significantly reduced

the content of tPA was significantly increased

the concentration of Ca

and the apoptosis in the cells were significantly reduced

CD34 expression increased in cells

and the protein expressions of VEGF

FAK and Paxillin were significantly increased (

0.05，

0.01).

Conclusion

CSF containing TQHXT protects BMECs from OGD/R injury possibly by promoting angiogenesis through the VEGF-VEGFR

/FAK/Paxillin signaling pathway.

关键词

通窍活血汤含药脑脊液氧糖剥夺/复糖复氧（OGD/R）脑微血管内皮细胞（BMECs）血管新生因子

Keywords

Tongqiao Huoxuetangcerebrospinal fluidoxygen-glucose deprivation/reoxygenationbrain microvascular endothelial cellsangiogenesis factor

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