Angiogenic And Neuroprotected Roles Of Erythropoietin Posttreatment On Neurovascular Unit In Oxygen Glucose Deprivation Injured Model: An In Vitro Study

Objective To study the angiogenic role of recombinant human erythropoietin (rH-EPO) on EA.hy 926 cells after oxygen and glucose deprivation followed by reoxygenation (OGD/R) in vitro. Methods Cells were subjected to OGD and stimulated by rH-EPO at the beginning of the reoxygenation procedure. MTT assay, Transwell assay and Matrigel assay were used to detect the angiogenic ability of cells post-OGD. The expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) mRNA and eNOS protein were also detected.Results OGD induced a significant decrease of proliferation, migration, and tube formation ability of EA.hy 926 cells and the expression of VEGF, eNOS mRNA and protein were significantly decreased. rH-EPO lessened the OGD-induced damage of proliferation, migration, and tube formation ability of cells and increased the expression of VEGF, eNOS mRNA and protein.Conclusion VEGF and eNOS probably play critical roles in the angiogenic effects of rH-EPO on cells post-OGD in vitro.Benificial effects and possible mechanisms of recombinant human erythropoietin posttreatment on neurovascular unit in OGD injured model: an in vitro studyObjective To study the benificial effects and possible mechanisms of recombinant human erythropoietin (rH-EPO) on neurovascular unit (NVU) after oxygen glucose (OGD) in vitro.Methods Three cell lines were cocultured to construct an NVU model in vitro. Transmission electron micronmicroscopy, biochemical method, fluorescence method et al.were used to test and validate it's structure and function. The model was subjected to OGD and stimulated by rH-EPO just at the beginning of the reoxygenation procedure. The apoptotic EA.hy 926 and SH-SY5Y cells stained with Annexin V-FITC/PI were mesured, and the oxidative (MDA)and antioxidative(SOD) indicators were detected too.The expression of inducible nitric oxide synthase(iNOS) protein and caspase-3 mRNA in the cytoplasm of SH-SY5Y were also detected.Results After coculturing for 7 days, the model presented the specific structure and function of BBB, e g. positiveγ-GT, tight junction and so on. OGD induced a significant increase in the apoptotic rate of EA.hy926 and SH-SY5Y, and the expression of iNOS and caspase-3 in the SH-SY5Y cell. Meanwhile, the oxidative and antioxidative system was in state of imbalance. However, rH-EPO could reverse the above mentioned damage.Conclution On the whole, the NVU model possess the BBB specific structure and function; posttreatment of NVU with rH-EPO at the beginning of reperfusion produced a significant reduction in the increased injury which caused by OGD.The possible mechanisms may include decreasing cell apoptosis , anti-free radical damage and regulating the expression of iNOS and caspase-3 and so on.