Experimental Study On The Angiogenesis Inhibition Effects Of Cinobufotalin In Human Umbilical Vein Endothelial Cells

Objective: Angiogenesis refers to the formation of new blood vessels from existing capillaries or veins behind capillaries,which generally includes the migration,proliferation and branching of endothelial cells to form vascular rings.In Ophthalmology,choroidal neovascularization(CNV)originated from the abnormal proliferation of choroidal capillaries is a pathological process in many fundus diseases and an important cause of vision loss.The pathogenesis of CNV is still unclear,and there are many problems in the clinical treatment.Cinobufotalin is a clinical anti-tumor drug,which can inhibit the proliferation and angiogenesis of tumor cells.Taking Human umbilical vein endothelial cells(HUVECs)as the objective,to investigate the effects of cinobufotalin on the proliferation,migration and lumen formation of HUVECs induced by VEGF in vitro,which will play the founder role for cinobufotalin application in the clinical treatment of CNV.Methods: HUVECs cells were divided into control group(only containing culture medium),VEGF group(simply adding 50ng/ml VEGF),VEGF+0.1μM cinobufotalin group(adding 50ng/ml VEGF and 0.1μM cinobufotalin),VEGF+0.2μM cinobufotalin group(adding 50ng/ml VEGF and 0.2μM cinobufotalin)and VEGF+0.5μM cinobufotalin group according to the different concentrations of cinobufotalin.CCK-8 assay was used to detect the effect of cinobufotalin on VEGF-induced HUVECs activity.Ed U assay was used to detect the impact of cinobufotalin on VEGF-induced HUVECs proliferation.Wound healing assay and Transwell assay were used to verify the effect of cinobufotalin on VEGF-induced HUVECs migration ability.The lumen formation assay was used to analyze the effect of cinobufotalin on VEGF-induced HUVECs angiogenesis formation.Results: 1.Compared with the control group,the cell activity,cell proliferation rate,horizontal migration rate,the number of vertically migrating cells,the number of branches and the length of branches formed in the VEGF group increased by 6.82%,6.1%,11.05%,1.24 times and 0.67 times,respectively,with statistical differences(P < 0.05).2.Compared with the VEGF group,the cell activity of VEGF+0.1μM cinobufotalin group,VEGF+0.2μM cinobufotalin group,and VEGF+0.5μM cinobufotalin group decreased by 16.34%,42.55% and 57.45%,respectively,with statistical differences(P=0.000,<0.001).Compared with the VEGF group,VEGF+0.1μM cinobufotalin group and VEGF+0.2μM cinobufotalin group decreased cell proliferation rate by 16.2%,19.48%,and horizontal migration rate by 11.94%,19.04%,respectively.The number of vertically migrated cells decreased by 55.34%,68.32%,and the number of branches formed by VEGF+0.1μM cinobufotalin group and VEGF+0.2μM cinobufotalin group decreased by 0.66 times and 1.21 times,respectively.The length of branches was reduced by 0.39 times and 0.76 times,with statistical differences(P < 0.05).Given the above data,cinobufotalin can inhibit the proliferation,migration,and vascular lumen formation of HUVECs induced by VEGF,and the higher the drug concentration,the stronger the inhibitory effect.Conclusions:1.Cinobufotalin can effectively inhibit the activity and proliferation of HUVECs induced by VEGF in a concentration-dependent manner.2.Cinobufotalin could inhibit the horizontal and vertical migration of HUVECs induced by VEGF,and the effect of inhibition is enhanced with the increase of cinobufotalin concentration.3.Cinobufotalin can inhibit the vascular lumen formation of HUVECs induced by VEGF and inhibit the length and number of vascular branches.The higher the concentration of cinobufotalin,the more potent the inhibition.4.Cinobufotalin has a potent inhibitory effect on angiogenesis,which is capable of implementing the subsequent experimental study of its application in the clinical treatment of CNV.