Effect Of Icariin On Angiogenesis And Mechanism In The Rat Model Of Hind Limb Ischemia

Objective: Explore the effects of icariin(ICA)on angiogenesis and mechanisms in the rat model of hind limb ischemia.Methods: In vivo,the unilateral femoral artery ligation of rats were used in this study.All of rats were randomly divided into control + ICA 40 mg/kg,model,model+ ICA 10 mg/kg,model + ICA 20 mg/kg and model + ICA 40 mg/kg.Histopathological alternations were observed by HE staining.Angiography was performed to observe the collateral circulation,the capillary density was used to detect the microvessels growth,the proliferation of endothelial cells was detected by immunohistochemistry,and the expression of VEGF,VEGFA,VEGFR2,Akt,e NOS,as well as the level of p-VEGFR2,p-Akt,p-e NOS by western blot.In vitro,the cell line HMEC-1 was used to observe the effect of ICA with or without PI3 K inhibitor(LY 294002)or e NOS inhibitor(L-NAME).The proliferation of HMEC-1 was examined by Brd U Elisa.The expression of VEGF,VEGF,VEGFR2,Akt,e NOS and the level ofp-VEGFR2,p-Akt,p-e NOS were detected by western blot.Results: In vivo results showed: compared with control group,the gastrocnemius muscle of model group showed ischemia-like alternations.Compared with model group,the ischemic damages of the ICA group were significantly improved.The results of angiography and immunohistochemistry indicated,compared with control group,model group showed some collateral vessel growth and endothelial cell proliferation,and microvascular growth was significantly reduced,and collateral circulation,microvessels growth and endothelial cell proliferation were significantly increased after administration of ICA in comparison with model group;western blot showed that compared with the control group,the expression of VEGF,VEGFA,p-VEGFR2,p-Akt,and p-e NOS in model group was significantly reduced,and the above protein levels were significantly increased at the dose of ICA 20 and 40 mg/kg.In vitro results showed: in the cell proliferation assay,Brd U Elisa showed ICA significantly increased the Brd U level compared with control group;western blot showed that compared with control group,the level of VEGF,p-VEGFR2,p-Akt and p-e NOS protein were increased in ICA groups.Both PI3 K inhibitor(LY 294002)and e NOS inhibitor(L-NAME)inhibited ICA-induced proliferation of HMEC-1.Conclusion: In this study,ICA promotes angiogenesis of hind limb ischemia rats and its mechanism is related to VEGF-mediated PI3K/Akt-e NOS signaling pathway.