丹酚酸A对氧糖剥夺诱导损伤的人脑微血管内皮细胞血管新生能力的保护作用及机制  

Protective effect of salvianolic acid A on angiogenesis of human brain microvascular endothelial cells injured by oxygen glucose deprivation and mechanisms

作　　者：张森 刘成娣 孔德文 蒋楠 孔令雷 杜冠华 ZHANG Sen;LIU Cheng-di;KONG De-wen;JIANG Nan;KONG Ling-lei;DU Guan-hua(Beijing Key Laboratory of Drug Target and Screening Research,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China;School of Pharmacy,Henan University,Kaifeng 475004,China)

机构地区：[1]中国医学科学院北京协和医学院药物研究所,药物靶点研究与新药筛选北京市重点实验室,北京100050 [2]河南大学药学院,河南开封475004

出　　处：《中国药理学与毒理学杂志》2022年第3期161-169,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(82004071);北京市自然科学基金(7182113);国家科技重大专项(2018ZX09711001-009-009)。

摘　　要：目的 探讨丹酚酸A(SAA)对血管内皮细胞低氧损伤后体外血管新生能力的作用及其机制。方法 采用氧糖剥夺(OGD)的方法构建人脑微血管内皮细胞(HBMEC)缺氧损伤模型,CCK-8法检测OGD2,4,6,8和10 h细胞存活率,确定OGD时间。HBMEC分为细胞对照组、OGD组、OGD+SAA 0.3,1.0和3.0μmol·L^(-1)组及OGD+依达拉奉10μmol·L^(-1)组,OGD 6 h后,用CCK-8法检测细胞存活率;Matrigel管腔形成实验观察OGD 2~10 h管腔形成;OGD 6 h后,检测管腔节点数、网眼数、分支数以及管腔长度;Ed U掺入实验检测细胞增殖,划痕实验检测细胞迁移距离。HBMEC分为细胞对照组、OGD组、OGD+SAA 3.0μmol·L^(-1)组和OGD+依达拉奉10μmol·L^(-1)组,OGD 6 h后,Western印迹实验检测低氧诱导因子1α(HIF-1α)、血管内皮生长因子A(VEGFA)及其受体VEGFR2蛋白表达水平及磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路蛋白磷酸化水平。结果 OGD 6 h细胞存活率为(56±6)%,确定为后续OGD时间。与细胞对照组相比,OGD组细胞存活率明显降低,形成管腔的节点数、网眼数、分支数以及管腔长度均显著减少(P<0.01,P<0.05),EdU阳性细胞比例和细胞迁移距离也明显下降(P<0.01)。与OGD组相比,OGD+SAA 3.0μmol·L^(-1)组和OGD+依达拉奉10μmol·L^(-1)组细胞存活率明显提高(P<0.05),形成管腔的节点数、网眼数、分支数以及管腔长度均显著升高(P<0.01,P<0.05),EdU阳性细胞比例和细胞迁移距离也明显增加(P<0.01,P<0.05);Western印迹结果显示,与OGD组相比,OGD+SAA 3.0μmol·L^(-1)组HIF-1α,VEGFA和VEGFR2蛋白表达显著增加(P<0.01,P<0.05),PI3K,Akt和mTOR磷酸化水平明显升高(P<0.05)。结论 SAA可能通过激活HIF-1α/VEGFA/VEGFR2及其下游信号通路PI3K/Akt/mTOR发挥内皮细胞保护和促进血管生成的作用。OBJECTIVE To investigate the effects of salvianolic acid A(SAA) on angiogenesis in vitro and the underlying mechanisms. METHODS A hypoxic-injury model for oxygen-glucose deprivation(OGD)-induced human brain microvascular endothelial cells(HBMECs) was used to investigate the effects of SAA on angiogenesis. CCK-8 assay was used to detect the cell viability at 0, 2, 4, 6, 8and 10 h after OGD to determine the OGD time. HBMECs were randomly divided into six groups: cell control, OGD, OGD+SAA 0.3, 1.0, 3.0 μmol·L^(-1), and OGD+edaravone 10 μmol·L^(-1) groups. After 6 h of OGD, cell viability was determined by CCK-8 assay. Matrigel tube formation assay was conducted to observe the formation of lumina 2-10 h after OGD. The numbers of nodes, meshes, branches and the lumen length were detected at 6 h. EdU incorporation assay was used to evaluate the cell proliferation ratio while cell scratch assay was used to detect the migration distance 6 h after OGD. HBMECs were divided into cell control, OGD, OGD+SAA 3.0 μmol·L^(-1) and OGD+edaravone 10 μmol·L^(-1) groups. The expressions of hypoxia inducible factor-1α(HIF-1α), vascular endothelial growth factor-A(VEGFA) and VEGF receptor-2(VEGFR2), and protein phosphorylation levels of phosphatidylinositol-3-kinase(PI3K), protein kinase B(Akt) and mammalian target of Rapamycin(mTOR) protein were determined by Western blotting 6 h after OGD. RESULTS The cell viability was(56±6)% 6 h after OGD, which was determined as the time of subsequent experiments. Compared with the cell control group, OGD resulted in a significant decrease in cell viability(P<0.01). SAA(3.0 μmol·L^(-1)) and edaravone(10 μmol·L^(-1))reversed OGD-induced cell injury and increased cell viability. In addition, SAA(3.0 μmol·L^(-1)) could significantly increase the numbers of nodes, meshes, branches, the lumen length in tube formation(P<0.05,P<0.01), the ratio of EdU-positive cells(P<0.01) and the migration distance(P<0.05), which were all reduced in the OGD group(P<0.05, P<0.01). Furthermore, SAA(3.0

关 键 词：丹酚酸A 脑微血管内皮细胞 血管新生 低氧诱导因子1Α 血管内皮生长因子A 

分 类 号：R285.5R[医药卫生—中药学] R966[医药卫生—中医学]