芒柄花素通过PI3K/Akt通路促进氧糖剥夺/再灌注损伤的大鼠脑微血管内皮细胞血管生成OA

[Objective]To investigate the effect of formononetin(FMNT)on angiogenesis in primary rat brain microvascular endothelial cells(RBMECs)injured by oxygen-glucose deprivation/reperfusion(OGD/R),and to preliminarily elucidate its possible molecular mechanism.[Methods]Primary RBMECs were isolated and cultured by enzymatic digestion combined with density gradient centrifugation,and subjected to OGD/R to establish injury cell model.Cells were divided into blank control(Control)group,model(OGD/R)group,FMNT low-dose(5 μmol·L-1),medium-dose(10 μmol·L-1)and high-dose(20 μmol·L-1)groups,as well as FMNT high-dose combined with phosphatidylinositol 3-kinase(PI3K)inhibitor LY294002 group.Cell viability was measured by cell counting Kit-8(CCK-8)assay,migration ability was evaluated by scratch wound assay,and angiogenic potential was analyzed by tube formation assay on Matrigel.Molecular docking of FMNT with PI3K and RAC-alpha serine/threonine-protein kinase(Akt1)protein was performed using AutoDock Vina 1.1.2 software to predict their binding affinities and interaction modes.The expression levels of PI3K,Akt and vascular endothelial growth factor(VEGF)were detected by Western blot.[Results]Compared with Control group,cell viability,migration and tube formation ability of RBMECs were significantly decreased in OGD/R group,accompanied with downregulated expression of phosphorylated-Akt(p-Akt)and VEGF protein(P<0.05,P<0.01).FMNT treatment alleviated these injuries,with FMNT high-dose group showing the most significant effects and upregulating p-Akt and VEGF protein expression(P<0.05,P<0.01).The PI3K inhibitor LY294002 significantly reversed the promoting effects of FMNT on cell viability,migration,tube formation and related protein expression.Molecular docking results showed that FMNT could spontaneously bind to both PI3K and Akt1 proteins,with binding energies of(-7.63±0.05)kcal/mol and(-7.80±0.00)kcal/mol,respectively,indicating good binding affinity.[Conclusion]FMNT ameliorates OGD/R-induced injury in RBMECs,significantly improves cell viability,and promotes migration and angiogenic capacity,with the mechanism closely associated with the activation of the PI3K/Akt signaling pathway and upregulation of VEGF expression.