| SMND-309 was a novel metabolite in rat brain after oral administration of salvianolic acid B(Sal B)and has been obtained by the in vitro degradation of Sal B.Previous studies in our laboratory have demonstrated that SMND-309 exhibits protective effects against cerebral ischemia,myocardial ischemia and liver fibrosis in rats.It is speculated that SMND-309 possessed the activity of anti-cerebral ischemia principally by promoting angiogenesis.However,there is no sufficient evidence proving the effects of SMND-309 on angiogenesis,especially at the cellular and molecular levels.The aim of this study was to investigate the angiogenic actions of SMND-309 on human umbilical vein endothelial cells(HUVEC)in vitro and on rats with focal cerebral ischemic reperfusion injury,and to elucidate the potential molecular mechanisms.In vitro experiments on HUVEC,the effects of SMND-309 on proliferation,adhesion,migration and capillary-like structure formation were examined;The changes in levels of erythropoietin(EPO)?EPO receptor(EPOR),phosphorylated EPOR(p-EPOR),signal transducer and activator of transcription 3(STAT3),phosphorylated STAT3(p-STAT3)and vascular endothelial growth factor(VEGF)in HUVEC treated with SMND-309 were detected;Knocking down EPOR gene and blocking tyrosine kinase Janus kinase 2(JAK2)pathways with inhibitor AG490 were employed to explore the potential molecular signaling pathways in SMND-309 nduced angiogenesis.On Sprague-Dawley rats with focal cerebral ischemic reperfusion injury nduced by middle cerebral artery occlusion(MCAO),the dose-effect,time-effect and effect of long-term administration of SMND-309 against cerebral ischemia were observed;The length and oranches of axon and the changes in levels of CD31,NeuN,EPO,EPOR,p-EPOR,STAT3,(?)STAT3 and VEGF in brain of MCAO rat treated with SMND-309 were determined;Celastrol,a inhibitor of VEGF receptor,and AG490 were used to explore the potential molecular signaling pathways in SMND-309 induced angiogenesis.The results showed that SMND-309 strongly induced the proliferation,adhesion to different extracellular matrix,migration and capillary-like structure formation,and enhanced the levels of p-EPOR,p-STAT3 and VEGF.SMND-309 improved the recovery of cerebral ischemia reperfusion injury in a dose-dependent manner and had a favourable therapeutic time-window(6 hr).SMND-309 still displayed a good effect against cerebral ischemia after a long-term administration.SMND-309 significantly promoted the expression of CD31 and VEGF,which were related to angiogenesis.SMND-309 also increased the survival of neuron,and increased the length and branches of axon.The above results showed that SMND-309 could reduce the apoptosis or necrosis of neuronal through promoting angiogenesis,and improve the recovery of neural structures and functions.Results from further experiments using gene silence of EPOR,AG490 or Celastrol showed that SMND-309 first activated EPOR and its downstream JAK2/STAT3,and p-STAT3 transferred into nucleus and binded to the promoter region of VEGF gene,which resulted in the up-regulation of VEGF level and promotion of angiogenesis.This study provided a new theoretical basis for the treatment of cerebral ischemic injury with SMND-309. |
