| Objective:To investigate the pharmacodynamic effect and mechanism of anti-tumor angiogenesis of Rehmannioside D and 3-hydroxy-4-methoxybenzoic acid,the active components of Veronica peregrina L.,by inhibiting endothelial mesenchymal transition（Endo MT）.Methods:Human umbilical vein endothelial cells（HUVECs）and human umbilical vein cell fusion cell（EA.hy926）were used to investigate the effects of Rehmannioside D and 3-hydroxy-4-methoxybenzoic acid on the two cells and compare them with the proteasome inhibitor MG132.1.The optimal dose of HUVECs and EA.hy926 cells was detected by Sulforhodamine B（SRB）.2.The effects of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 on angiogenesis in vitro were assessed by tubule formation test and rat aortic ring angiogenesis test.3.The effects of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 on the cell longitudinal migration and invasion ability of HUVECs and EA.hy926 cells were assessed by the transwell chamber method.4.The effects of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 on the cell lateral migration and invasion ability of HUVECs and EA.hy926 cells were assessed using the Oris TMcell migration and invasion kit.5.The effects of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 on HUVECs and EA.hy926 cytoskeleton arrangement was assessed by F-actin staining.6.Cellular immunofluorescence experiment was involved to assess the effects of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 on the localization and expression of Vimentin,VE-cadherin,Runx2 in HUVECs and EA.hy926 cells.7.Western Blotting assay was involved to assess the effects of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 on the expression of Snail,phosphorylatedSnail（p-Snail）,Slug,phosphorylatedSlug（p-Slug）,Runx2,phosphorylated Runx2（p-Runx2）in HUVECs and EA.hy926 cells.Result:1.To determine the concentration of HUVECs cells,Rehmannioside D（final concentrations of 0.625,1.25,2.5μmol/L）,3-hydroxy-4-methoxybenzoic acid（final concentrations of 12.5,25,50μmol/L）,MG132（final concentrations of 0.625,1.25,2.5μmol/L）;The concentration of drug action in the EA.hy926 cells,Rehmannioside D（final concentrations of 0.3125,0.625,1.25μmol/L）,3-hydroxy-4-methoxybenzoic acid（final concentrations of 12.5,25,50μmol/L）and MG132（final concentrations of1.25,2.5 and 5μmol/L）.2.The results of tubule formation experiment showed that compared with the control group,the number of HUVECs and EA.hy926 cells treated with Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 was lower than that of the control group.3.The results of angiogenesis in the rat aortic ring showed that compared with the control group,the number of neovascularization and cell number in vitro after the treatment of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid and MG132 were lower than that of the control group.4.The results of transwell cell method showed that compared with the control group,HUVECs and EA.hy926 cells were treated with different doses of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 respectively,and the number of migrating and invading cells were lower than that of the control group,with statistical difference（P<0.01）.5.The results of Oris TMcell migration and invasion experiment showed that compared with the control group,each dose group of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 could inhibit the invasion of HUVECs and EA.hy926 cells to the central region,and the size of the central cell-free region was statistically different（P<0.01）.6.The F-actin staining method showed that in the control group of cells,F-actin was neatly arranged along the long axis of the cell and accumulated into stress fibers in the cell periphery.However,F-actin in the cells of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 showed a dose dependent disorder,suggesting that Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132could significantly inhibit the arrangement of HUVECs and EA.hy926 cell cytoskeleton.7.The results of cell immunofluorescence test showed that compared with the control group,the expression of Vimentin and Runx2 protein in HUVECs and EA.hy926 cells in each dose group of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132were down regulated,while the expression of VE-cadherin protein was up regulated.8.The results of Western blotting showed that the expression of p-Snail,p-Slug and p-Runx2 protein in HUVECs cells in each dose group of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 was significantly lower than that in the control group,and there was a statistical difference（P<0.01）,but there was no significant effect on the expression of Snail,Slug and Runx2 protein;The expression of p-Snail,p-Slug and p-Runx2 protein in EA.hy926 cells in different dose groups of Rehmannioside D,3-hydroxy-4-methoxybenzoic acid,MG132 were significantly decreased,with statistical difference（P<0.01）,but there was no significant effect on the expression of Snail,Slug and Runx2 protein.Conclusion:1.The active components of Veronica peregrina L.,Rehmannioside D,3-hydroxy-4-methoxybenzoic acid and proteasome inhibitor MG132,all significantly inhibit the migration,invasion and angiogenesis of vascular endothelial cells,and have the ability to significantly inhibit angiogenesis of vascular endothelial cells.2.Inhibition of the activation of key proteins of endothelial mesenchymal transformation and the migration phenotype and ability of mesenchymal cells,then inhibiting endothelial interstitial transformation in vascular endothelial cells may be one of the anti-tumor angiogenic mechanisms of Rehmannioside D and3-hydroxy-4-methoxybenzoic acid.3.The proteasome inhibitor MG132 has similar pharmacological results with Rehmannioside D and 3-hydroxy-4-methoxybenzoic acid in cell motility test,tubulation test,morphology test and Western Blotting assay,suggesting that MG132 may have the mechanism of inhibiting the endothelial mesenchymal transition. |
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