Study On The Effects And Mechanisms Of Flavonoids From The Leaves Of Carya Cathayensis Sarg.Inhibiting Angiogenesis

Objective:This study aims to investigate the inhibitory effect of total flavonoid(TF)which was isolated from the leaves of Carya cathayensis Sarg.(LCC)and five flavonoids,i.e.cardamonin,pinostrobin chalcone(PC),wogonin,chrysin and pinocembrin that were the main components of the TF on angiogenesis,and the preliminary mechanisms.The strongest component was screened.Then,explore the role of miR-21 in the element on anti-angiogenesis by building cell model and using miR-21 as a target.This experiment provides a new therapeutic target for vascular disease.Method:(1)Cultivate HUVECs in vitro to built the cell growth curve.The proliferation of HUVECs induced by vascular endothelial growth factor(VEGF)was measured by cell counting.(2)The anti-angiogenic activities of flavonoids from LCC on VEGF induced angiogenesis were measured by mouse aortic ring.(3)The effect of flavonoids from LCC on proliferation and migration of HUVECs induced by VEGF was respectively detected by cell counting and wound healing assay,and the expression of p-ERK1/2 and p-AKT of HUVECs by Western blotting.(4)In order to screen the miRNA of the most obvious change,we employ SYBR PCR to detect the effect of cardamonin on the expressions of miRNAs of HUVECs induced by VEGF in different times,for example,miR-16,miR-21 and miR-23a.(5)Employ SYBR PCR to detect the expression of miR-21 after transfected by miR-21 inhibitor and mimic.Then,the proliferation and migration of HUVECs treated with cardamonin after induced by VEGF was respectively detected by MTS and wound healing assay,the effect of cardamonin on angiogenesis by tube formation in vitro.Results:(1)Take use of 2×104/mL HUVECs cultured for 24 h and changed liquid according to HUVECs growth curve.The proliferation of HUVECs was significantly increased(p<0.01)with 8 ng/mL VEGF and in a concentration-dependent manner at 12 h and 24 h(p<0.01),the most significantly at 24 h,while there was no obvious change with 4 ng/mL and 10 ng/mL VEGF(p>0.05).(2)Compared with VEGF alone,the TF and five pure compounds remarkably suppressed angiogenesis after cultured aortic ring for 14 d,and cardamonin showed the best effect among them,while PC presented the weakest anti-angiogenic activity.(3)The TF,cardamonin,pinocembrin,and chrysin obviously suppressed both VEGF-induced HUVECs proliferation and migration.However,PC and wogonin slightly inhibited VEGF-induced proliferation but also remarkably suppressed those of migration in HUVECs.Our further study showed that cardamonin decreased the phosphorylation of ERK and AKT induced by VEGF with a dose-dependent manner in HUVECs.(4)SYBR PCR results indicated that miR-21 change was more obvious in different times treated with cardomonin.(5)The expression of miR-21 was respectively decreased and increased after transfected by miR-21 inhibitor and mimic for 24 h.F urthermore,compared with the cardomonin group,the miR-21 inhibitor group remarkly inhibited HUVECs proliferation,migration and tube formation with 50 ?M cardomonin,while the miR-21 minic group remarkly promoted HUVECs proliferation,migration and tube formation with 50 ?M cardomonin.Conclusion:(1)All the flavonoids tested in our experiments present angiogenic effects and cardamonin showed the best effect among them.(2)Cardamonin treated HUVECs exhibites a decreased level of p-ERK1/2 and p-AKT with a dose-depedent manner,indicating ERK 1/2 and AKT signaling might be,at least in part,involved in this process.(3)MiR-21 change shows more obvious among the miRNAs in different times treated with cardomonin that miR-21 may be involved in cardamonin-mediated angiogenesis inhibition.