通窍活血汤含药脑脊液对OGD/R损伤大鼠BMECs的保护作用

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(10μmol·L^-1),卡博替尼(BMS)组(1μmol·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(P<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(P<0.05,P<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(R2)/FAK/Paxillin信号通路促进血管生成来发挥作用。

Protective Effect of Cerebrospinal Fluid Containing Tongqiao Huoxuetang on Brain Microvascular Endothelial Cells from Rats with Oxygen Glucose Deprivation/Reoxygenation Injury

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(10μmol·L-1),the cabozanix group(1μmol·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 Ca2+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 Ca2+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(P<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 Ca2+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(P<0.05,P<0.01).Conclusion:CSF containing TQHXT protects BMECs from OGD/R injury possibly by promoting angiogenesis through the VEGF-VEGFR2/FAK/Paxillin signaling pathway.