Mechanism of astragaloside Ⅳ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification
Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ)combined with Panax notoginseng saponins(PNS)in regulating angiogenesis to treat cerebral ischemia.The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis.In vivo experiment:SD rats were randomized into sham,model,and AST Ⅳ(10 mg·kg-1)+PNS(25 mg·kg-1)groups,and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO)method.AST Ⅳ and PNS were administered by gavage twice a day.the Longa method was employed to measure the neurological deficits.The brain tissue was stained with hematoxylin-eosin(HE)to reveal the pathological damage.Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF),and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA).Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2),VEGFA,phosphorylated phosphatidylinositol 3-kinase(p-PI3K),and phosphorylated protein kinase B(p-AKT)in the brain tissue.In vitro experiment:the primary generation of rat brain microvascular endothelial cells(rBEMCs)was cultured and identified.The third-generation rBMECs were assigned into control,model,AST Ⅳ(50 μmol·L-1)+PNS(3 0μmol·L-1),LY294002(PI3K/AKT signaling pathway inhibitor),740Y-P(PI3K/AKT signaling pathway agonist),AST Ⅳ+PNS+LY294002,and AST Ⅳ+PNS+740Y-P groups.Oxygen glucose deprivation/re-oxygenation(OGD/R)was employed to establish the cell model of cerebral ischemia-reperfusion injury.The cell counting kit-8(CCK-8)and scratch assay were employed to examine the survival and migration of rBEMCs,respectively.Matrigel was used to evaluate the tube formation from rBEMCs.The Transwell assay was employed to examine endothelial cell permeability.Western blot was employed to determine the expression of VEGFR2,VEGFA,p-PI3K,and p-AKT in rBEMCs.The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction.Most of these 21 targets were involved in the PI3K/AKT signaling pathway.The in vivo experiments showed that compared with the model group,AST Ⅳ+PNS reduced the neurological deficit score(P＜0.05)and the cell damage rate in the brain tissue(P＜0.05),promoted the expression of vWF and VEGFA(P＜0.01)and angiogenesis,and up-regulated the expression of proteins in the PI3K/AKT pathway(P＜0.05,P＜0.01).The in vitro experiments showed that compared with the model group,the AST Ⅳ+PNS,740Y-P,AST Ⅳ+PNS+LY294002,and AST Ⅳ+PNS+740Y-P improved the survival of rBEMCs after OGD/R,enhanced the migration of rBEMCs,increased the tubes formed by rBEMCs,up-regulated the expression of proteins in the PI3K/AKT pathway,and reduced endothelial cell permeability(P＜0.05,P＜0.01).Compared with the LY294002 group,the AST Ⅳ+PNS+LY294002 group showed increased survival rate,migration rate,and number of tubes,up-regulated expression of proteins in the PI3K/AKT pathway,and decreased endothelial cell permeability(P＜0.05,P＜0.01).Compared with the AST Ⅳ+PNS and 740Y-P groups,the AST Ⅳ+PNS+740Y-P group presented increased survival rate,migration rate,and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway,and reduced endothelial cell permeability(P＜0.01).This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.
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