Hydroxysafflor Yellow A Regulates Angiogenesis To Protect Against Oxygen Glucose Deprivation/Reoxygenation Injury Of Cerebral Microvascular Endothelial Cells In Rats By SIRT1-HIF-1?-VEGFA Signal Transduction Pathway

ObjectiveTo study the effects and possible mechanisms of hydroxysafflor yellow A?HSYA?on oxygen-glucose deprivation/reoxygenation?OGD/R?injured brain microvascular endothelial cells?BMECs?via SIRT1-HIF-1?-VEGFA signaling pathway.Methods1.Regulation of hydroxysafflor yellow A?HSYA?on angiogenesis of brain microvascular endothelial cells?BMECs?injured by oxygen-glucose deprivation/reoxygenation?OGD/R?1.1 Primary separation,culture and identification of BMECs?1?Primary BMECs are extracted by enzymatic digestion and cultured routinely;?2?Observed the cell morphology by light microscope and identified BMECs by immunofluorescence staining of?factor related antigen;1.2 Establishment of OGD/R modelAfter the third generation BMECs were rinsed twice with Phosphate buffer solution?PBS?,the glucose-free medium was added,and it was placed in a pre-adjusted37?constant temperature hypoxia chamber?95%N₂,5%CO₂?for 2 h.Then the glucose-free medium was replaced by a high-glucose medium,and the OGD/R model was established by reoxygenated for 24 h in a constant temperature incubator at 37?and 5%CO₂;1.3 The levels of vascular endothelial growth factor?VEGF?,angiopoietin?Ang?and platelet-derived growth factor?PDGF?in the cell supernatant were determined by enzyme-linked immunosorbent assay?ELISA?;1.4 The expression of VEGFA,Ang-2 and PDGFB proteins in cells were detected by western-blotting;1.5 Cell proliferation was detected by CCK-8 method;1.6 Cell adhesion performance was detected with cell adhesion detection kit;1.7 The expressions of CD34 and CD31 in cells were detected by immunofluorescence staining;1.8 Cell migration was detected by wound healing test;1.9 Cell migration was detected by Transwell chamber.2.Mechanisms of HSYA on angiogenesis regulation of BMECs injured by OGD/R2.1 The m RNA levels of SIRT1,HIF-1?and VEGFA in cells were detected by Real-Time PCR;2.2 The expressions of SIRT1,HIF-1?and VEGFA proteins in cells were detected by western-blotting.Results1.HSYA can regulate the angiogenesis of BMECs with OGD/R injury?1?The morphological observation of the cells showed that the extracted primary cells began to take on a long spindle shape on the 6th day,and continued to culture on the10th day,and the cells showed paving stones;the results of immunofluorescence staining of?factor related antigen showed that the extracted primary cells emitted significant green fluorescence in the cytoplasm,and the nucleus was counterstained with blue by DAPI;?2?ELISA test results showed that after OGD/R modeling,the levels of VEGF,Ang and PDGF in the cell supernatant increased,while HSYA could further increase the levels of VEGF,Ang and PDGF in the cell supernatant;?3?Western-blotting results showed that after OGD/R modeling,the expression of VEGFA,ang-2 and PDGFB proteins in cells increased,while HSYA could further increase the expression of VEGFA,ang-2 and PDGFB proteins.?4?CCK-8 assay showed that OGD/R reduced cell viability,while HSYA significantly increased the cell proliferation of BMECs induced by OGD/R;?5?Cell adhesion test results showed that OGD/R reduced the cell adhesion performance,while HSYA significantly increased the cell adhesion performance of BMECs induced by OGD/R;?6?The results of cell immunofluorescence staining showed that compared with the normal control group,CD34 and CD31 factors were expressed in the OGD/R group,while CD34 and CD31 factors were significantly increased in the HSYA group;?7?The results of wound healing test and Transwell chamber migration experiment showed that HSYA could significantly increase the cell migration of BMECs induced by OGD/R.2.HSYA regulates angiogenesis of BMECs induced by OGD/R through SIRT1-HIF-1?-VEGFA signal transduction pathway?1?Real-Time PCR results showed that,compared with the normal control group,the relative expression of SIRT1 m RNA in the OGD/R group was significantly decreased,while the relative expression of HIF-1?and VEGFA m RNA were significantly increased.Compared with the OGD/R group,the relative expression levels of SIRT1,HIF-1?,and VEGFA m RNA in the HSYA group were significantly increased.Compared with the HSYA group,the relative expressions of SIRT1,HIF-1?and VEGFA m RNA were significantly decreased when the inhibitor EX-527 was combined with HSYA.When the agonist Resveratrol?RSV?was combined with HSYA,the relative expression levels of SIRT1,HIF-1?and VEGFA m RNA were significantly increased;?2?Western-blotting results showed that,compared with the normal control group,the relative expression of SIRT1 protein in the OGD/R group was significantly reduced,while the relative expression of HIF-1?and VEGFA proteins were significantly increased.Compared with the OGD/R group,the relative expression levels of SIRT1,HIF-1?,and VEGFA proteins in HSYA group were significantly increased.Compared with the HSYA group,the relative expression levels of SIRT1,HIF-1?,and VEGFA proteins were significantly decreased when the inhibitor EX-527 was combined with HSYA.When the agonist RSV was combined with HSYA,the relative expression levels of SIRT1,HIF-1?,and VEGFA proteins were significantly increased.ConclusionCombined with the above experimental results,HSYA has a regulatory effect on the angiogenesis of BMECs induced by OGD/R,which may promote angiogenesis and play an anti-injury effect through the SIRT1-HIF-1?-VEGFA signal transduction pathway.