| 摘要: |
| 目的：探讨芍药苷（paeoniflorin， PF）对氧糖剥夺（oxygen-glucose deprivation， OGD）损伤的
脑微血管内皮细胞（brain microvascular endothelial cells，BMECs）血管新生的影响，并结合网络药理学与实
验验证阐明其潜在分子机制。方法：采用OGD 方法构建BMECs 损伤模型，随机分为模型（OGD）组、PF 低
剂量（PF-L）组（OGD+10μmol/L PF）、PF 中剂量（PF-M）组（OGD+20μmol/L PF）、PF 高剂量（PF-H）组
（OGD+40μmol/L PF），阳性药物丁苯酞（3-n-butylphthalide，NBP）组（OGD+10μmol/L NBP），以正常未处理
细胞作为对照（control）组。细胞计数试剂盒 - 8（cell counting kit-8，CCK-8）法检测PF 对BMECs 的毒性作
用及细胞增殖情况；划痕愈合实验检测细胞迁移能力；成管实验检测细胞血管形成能力；免疫荧光染色法检测
血管内皮生长因子（vascular endothelial growth factor，VEGF）、血小板内皮细胞黏附分子（platelet endothelial cell
adhesion molecule-1 ，PECAM-1/CD31）的表达水平；网络药理学方法筛选PF 作用于缺血性脑卒中（ischemic
stroke，IS）相关血管新生的潜在靶点及信号通路；分子对接预测PF 与磷脂酰肌醇3- 激酶（phosphatidylinositol
3-kinase，PI3K）、蛋白激酶B（protein kinase B，AKT）蛋白的结合活性；蛋白质印迹法（western blot，WB）检
测PI3K、AKT、磷酸化磷脂酰肌醇3- 激酶（phosphorylated phosphatidylinositol 3- kinase，p-PI3K）、磷酸化蛋白
激酶B（phosphorylated protein kinase B，p-AKT）蛋白表达。结果：CCK-8 实验结果显示，一定浓度范围内的
PF 对BMECs 无明显毒性，且筛选出其促进OGD 损伤后BMECs 增殖的最佳浓度为20 μmol/L ；与模型组相
比，PF 中、高剂量组及NBP 组均能显著促进BMECs 划痕愈合率、增加成管数量，上调VEGF、CD31 的表达，增
强PI3K/AKT 通路相关蛋白表达，差异均有统计学意义（P <0.01）。网络药理学筛选获得PF 调控IS 相关脑血
管新生的131 个交集靶点；京都基因与基因组数据库（kyoto encyclopedia of genes and genomes， KEGG）通路富
集分析结果显示，靶点显著富集于PI3K/AKT、丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）
及脂质与动脉粥样硬化等信号通路。分子对接结果显示，PF 与PI3K、AKT 均具有良好的结合活性。结论：PF
可促进OGD 损伤后BMECs 的血管新生，其机制可能是通过激活PI3K/AKT 信号通路发挥作用。 |
| 关键词: 芍药苷 血管新生 脑微血管内皮细胞 缺血性脑卒中 PI3K/AKT 通路 |
| DOI： |
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| Mechanism of action of paeoniflorin in repairing brain microvascular endothelial injury: A studybased on network pharmacology and experimental research |
| DUAN Junxia,ZHAO Yaqing,XIONG Tingting,GUO Yurou,LU Jin,DING Huang,ZHANG Wei |
| (School of Integrated Traditional Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha 410208, Hunan, China;Key Laboratory of Hunan Province for Prevention and Treatment of Integrated Traditional Chinese and Western Medicine on Cardiocerebral Diseases, Changsha 410208, Hunan, China) |
| Abstract: |
| Objective: To investigate the effect of paeoniflorin （PF） on angiogenesis in brain microvascular endothelial
cells （BMECs） injured by oxygen-glucose deprivation （OGD）, and to clarify its potential molecular mechanism
based on network pharmacology and experimental validation. Methods: The OGD method was used to establish a model
of BMEC injury, and then the cells were randomly divided into model group （OGD）, low-dose PF group （PF-L group
treated with OGD+10 μmol/L PF）, middle-dose PF group （PF-M group treated with OGD+20 μmol/L PF）, highdose
PF group （PF-H group treated with OGD+40 μmol/L PF）, and positive drug 3-n-butylphthalide （NBP） group
（OGD+10 μmol/L NBP）, with normal untreated cells included as the control group. CCK-8 assay was used to observe
the toxic effect of PF on BMECs and cell proliferation; wound healing assay was used to observe cell migration ability; tube
formation assay was used to observe the angiogenic ability of the cells; immunofluorescent staining was used to measure the
expression levels of vascular endothelial growth factor （VEGF） and platelet endothelial cell adhesion molecule-1 （PECAM-
1/CD31）. Network pharmacology methods were used to identify the potential targets and signaling pathways of PF
acting on angiogenesis related to ischemic stroke （IS）; molecular docking was used to predict the binding activity of PF
to phosphatidylinositol 3-kinase （PI3K） and protein kinase B （AKT）; Western blot was used to measure the protein
expression levels of PI3K, AKT, phosphorylated PI3K （p-PI3K）, and phosphorylated AKT （p-AKT）. Results: CCK-
8 assay showed that PF within a certain concentration range had no obvious toxicity on BMECs, and 20 μmol/L was determined
as the optimal concentration promoting the proliferation of BMECs after OGD injury. Compared with the model group,
the middle- and high-dose PF groups and the NBP group significantly promoted wound healing rate, increased the number
of tubes formed, upregulated the expression levels of VEGF and CD31, and enhanced the expression levels of the proteins
related to the PI3K/AKT pathway （P < 0.01）. Network pharmacology obtained 131 intersecting targets for PF in regulating
IS-related angiogenesis, and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that
the targets were significantly enriched in the signaling pathways including PI3K/AKT, mitogen-activated protein kinase,
and lipid and atherosclerosis. Molecular docking showed that PF had good binding activity to both PI3K and AKT. Conclusion:
PF can promote angiogenesis in BMECs after OGD injury, possibly by activating the PI3K/AKT signaling pathway. |
| Key words: paeoniflorin angiogenesis brain microvascular endothelial cells ischemic stroke phosphatidylinositol
3-kinase/protein kinase B pathway |
