Hydroxysafflor Yellow A Regulates Angiogenesis To Protect Against Oxygen Glucose Deprivation/Reoxygenation Injury Of Cerebral Microvascular Endothelial Cells In Rats By SIRT1-HIF-1α-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-1α-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 ofⅧfactor 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-1αand VEGFA in cells were detected by Real-Time PCR;2.2 The expressions of SIRT1,HIF-1αand 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 ofⅧfactor 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-1α-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-1αand VEGFA m RNA were significantly increased.Compared with the OGD/R group,the relative expression levels of SIRT1,HIF-1α,and VEGFA m RNA in the HSYA group were significantly increased.Compared with the HSYA group,the relative expressions of SIRT1,HIF-1αand 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-1αand 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-1αand VEGFA proteins were significantly increased.Compared with the OGD/R group,the relative expression levels of SIRT1,HIF-1α,and VEGFA proteins in HSYA group were significantly increased.Compared with the HSYA group,the relative expression levels of SIRT1,HIF-1α,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-1α,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-1α-VEGFA signal transduction pathway.