Effect Of Gambogenic Acid On HUVEC Angiogenesis By PI3K/AKT/VEGF/eNOS Signaling Pathway

By previous studies,we found that the GNA showed good biological activity in inhibiting tumor angiogenesis,the concentration of GNA above 2.0μM can block blood vessels in conditioned medium to form factor effect on promoting proliferation of HUVEC,and in the certain concentration range are dose dependent.Composite will be two different types of cells to develop,the results show that concentration at 2.0μM above GNA can block induced A549 lung cancer cell culture supernatant of tubular form,the prompt GNA can prevent tumor cells secrete angiogenic factors,influence the formation of cell proliferation and lumen,become an important part of the intervention in tumor angiogenesis.So the study of how the GNA intervention angiogenesis mechanism,as the development of anti-cancer drugs,for its theoretical basis.Objective:Set up non direct contact with the cell co-culture system,in vitro angiogenesis,the process of the evaluation of GNA effects on angiogenesis.Study the effects of GNA signal transduction mechanism of angiogenesis,clearing PI3K/Akt/VEGF/eNOS signaling pathways in the role of the effect,in order to further study the key targets of its inhibition of angiogenesis.Methods:In this study,we cultivate the human umbilical vein endothelial cells;Determined by MTT method and cell clone test method,the GNA effect on HUVEC cell proliferation;Morphological changes were observed under inverted microscope HUVEC cells;The DAPI staining and Annexin V-FITC/PI double dye the detection of human umbilical vein endothelial cell apoptosis;Apply a thin layer of collagen vascular endothelial cell of two-dimensional culture model is set up,watch GNA effects on the phenomenon of HUVEC into tube;GNA scratches healing migration experiment at different concentrations on HUVEC cell migration change;GNA Transwell migration experiment at different concentrations on HUVEC cell migration change;Western blot method measuring GNA on tumor angiogenesis of eNOS,VEGF,PI3K,and AKT and PTEN protein;By adding LY294002 to HUVEC cells to study the mechanism of action of GNA may be;After using RNA interference means to inhibit the expression of PTEN gene,using RT-PCR method to observe the GNA of PI3K,and AKT and VEGF mRNA.Results:(1)Respectively by determined by MTT method and the clone forming experiments,GNA can significantly inhibit the proliferation of HUVEC cells,and with the GNA concentration and time of treatment,significantly decreased the cell survival rate.(2)Through the microscope,and the control group,while increasing the GNA concentration has significant damage to the cellular structure,along with the bubble burst,even apoptosis.(3)GNA on HUVEC cell morphological changes under the fluorescence microscope;Blank control group,after DAPI staining nucleus normal uniform light blue,blue and white cells appeared apoptosis,several nuclear fragmentation,have obvious characteristics of apoptosis.(4)The test results show that the effect of GNA on the HUVEC cells,with the increase of drug concentration,cell apoptosis rate increased significantly.(5)Compared with the blank control group,conditioned medium of each treatment group cells into tube number decreases,and a dose dependent.Can significantly reduce the tube samples,suggesting that GNA prevent HUVEC cells form a lumen.(6)Between groups within 24 h of scratches to observe cells.Results show that each cell 0 h,there was no significant difference in each treatment group cells,compared with the blank group scratches healing slowly,and through the small chamber transwell cell number is reduced,suggests GNA can inhibit HUVEC scratches healing,and present a dose dependent.(7)With the GNA dosage increasing expression of VEGF and eNOS drop;PTEN protein expression as the GNA dose increases with the increase of p-PI3K and p-Akt protein expression showed a trend of decreasing;the change trend of PI3K,and Akt protein is not obvious.(8)Inhibitors in advance after processing,the GNA and inhibitor can effectively inhibit eNO production and cell migration induced by VEGF,VEGF and eNOS and iNOS protein expression showed a trend of lower,and the combined effect is better.(9)RT-PCR detection shows that PI3K,and AKT and VEGF mRNA gene expression can be raised after PTEN-siRNA transfecting into the cell.Conclusion:GNA can regulate PI3K/Akt/VEGF/eNOS signaling pathways,decreasing related proteins and gene expression,inhibiting the angiogenesis of HUVEC cells.