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

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 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.