Experimental Study On Angiogenic Effect And Its Mechanism Of Total Flavone Of Abelmoschus Manihot

Abelmoschus Manihot is "an important drug in treatment of various malignant sore, cellulites and burns ". Clinical effects are significant in topical treatment of chronic ulcers. Modern pharmacological research also found that Total Flavone of Abelmoschus Manihot as active ingredients has a significantly protective effect on myocardial and cerebral ischemia. The thesis is a study on angiogenic effect and some molecular mechanisms of TFA,and the main contents are the following sections:PartⅠ:TheorySummary and elaboration on the angiogenesis process and the adjustment mechanism, the progress of therapeutic angiogenesis, Chinese Medicine Research on angiogenesis, clinical and basic research on Abelmoschus Manihot and TFA are reviewed. Angiogenesis is from endothelial cells migration, through proliferation, migration and differentiation, to lumen formation, vessel maturation and stability, and the process have a variety of factors such as VEGF(vascular endothelial growth factor)which is the most important. Promoting angiogenesis treatment in the disease at present mainly focused on ischemic diseases and tissue engineering, and many kinds of inducer or gene therapy by factors related with angiogenesis are used. Results of animal models and clinical tset be recognized. Chinese medicine treatment of ischemic disease is effective,and has a long history, rich clinical experience, and basic experimental angiogenesis research has been started. TFA shows a good protection to heart and brain ischemic injury in animal models.PartⅡ: Experimental Study1. Effects of TFA on proliferation, migration and tube formation in human umbilical vein endothelial cell. Used MTT to test proliferation of human umbilical vein endothelial cell in different concentrations of TFA, effective concentrations and time selected. Used flow cytometry to observe effects of TFA on human umbilical vein endothelial cells in cell cycle. Used transwell small room, in vitro tube formation to observe effects of the drugs on human umbilical vein endothelial cells in migration and the vascular capacity impact. Results: After 72h, group TFA5μg/ml, 10μg/ml,20μg/ml was higher absorbance, as compared with the control group, significant difference (p=0.002,0.000,0.050). HUVEC cells in G0-G1 phase was significantly reduced, S phase fraction increased significantly, among the four groups the difference of proportion of cells in each cycle was significant (p= 0.000).5,10,20μg/ml TFA increased the migration numbe of human umbilical vein endothelial cell, and compared with the control group, the difference was significant (p=0.000,0.000,0.028), in which 10μg/ml TFA made the largest number of cell migration, followed by group 5μg/ml and 20μg/ml. In group 10μg/ml TFA, role of people umbilical vein endothelial cell tube formation was significantly increased compared with the control, followed by 20μg/ml concentration group, the difference was statistically significant (p=0.000,0.013), but group 5μg/mlTFA compared with the control, the difference was not significant (p= 0.096). Conclusion: 5-20μg/ml TFA promote the proliferation of human umbilical vein endothelial cells significantly after72h, enabling more cells into the proliferative phase, and can promote cell migration.10-20μg/mlTFA can promote tube-like structure formation of human umbilical vein endothelial cells.2. Effects of TFA on apoptosis and fas, bcl-2 in HUVEC. Application of flow cytometry on human umbilical vein endothelial cell apoptosis and protein fas. Immunocytochemical staining and Western blotting on human umbilical vein vascular endothelial cell apoptosis protein bcl-2 effects. Results:On TFA, after 72h, of Human umbilical vein endothelial cells, the apoptosis rate was 10.1% in control group,7.2% in group TFA5μg/ml,3.9%, in group TFA10μg/ml,8.5% in group TFA 20μg/ml. Compared with the control group, difference was significant (p<0.01).5μg/ml, 10μg/ml,20μg/ml TFA made fas expression level decreased in umbilical vein endothelial cells, compared with the control group, significant difference (p= 0.000,0.000,0.028). Compared with control group, bcl-2 expression was slightly higher in group TFA 5μg/ml, the difference was not statistically significant (p= 0.075). In 10,20μg/ml group the expression of bcl-2 was statistically significant difference (p= 0.000), but significantly lower than the control group. Conclusion: 5-20μg/ml TFA can inhibit HUVEC apoptosis, reduc the expression of protein fas which is likely to the mechanism of apoptosis. TFA can not increase the apoptosis-related protein bcl-2 expression in HUVEC.3. Effects of TFA on VEGF/KDR expression in human umbilical vein endothelial cells. Immunocytochemical staining and Western blotting on vascular endothelial growth factor and its receptor KDR protein expression in human umbilical vein endothelial cells. Results:The expression of VEGF were strongly positive in HUVEC, especially in group 10μg/ml, cytoplasm staining were significantly enhanced at the depth and scope. Group 5,10,20μg/ml TFA compared with the control group, p values were 0.010,0.000,0.005. The expression of KDR were strongly positive in the cytoplasm and membrane staining were significantly enhanced at the depth and scope in group TFAlOμg/ml, 10μg/ml TFA significantly enhanced the expression of KDR in HUVEC, compared with control group, p= 0.007, and compared with the control group, in group 5,20μg/ml TFA, expression of KDR had no significant change (p> 0.05). Conclusions: TFA promote angiogenesis by increasing the expression of VEGF in cells and maybe increase the expression of KDR also.4. Effects of TFA on the chick chorioallantoic membrane (CAM) angiogenesis. Used chicken chorioallantoic membrane model to observe CAM angiogenesis. Results:The blood vessels presented the radial growth around carriers as center, significantly in group TFAlOμg/ml, and continued to branch out multi-stage small vessels, large vessels increased significantly. The number of CAM blood vessels increased also in group 5-20μg/mlTFA, the difference was statistically significant.Compared with control group, in TFA5,10,20μg/ml group, at number of blood vessels away from the carrier edge lmm, p values were 0.000,0.000,0.000, and 5mm away from the edge, p= 0.000,0.000,0.000. Conclusion: 5-20μg/ml TFA can promote the chick embryo chorioallantoic membrane angiogenesis, indicating its could promote angiogenesis in vivo.This study shows that TFA promote angiogenesis obviously, encourage endothelial cell proliferation, migration and tube-like structure formation, and inhibit vascular endothelial cell apoptosis. The mechanism is mainly regulated VEGF expression in vascular endothelial cells, and down regulation of fas protein expression. Specified concentration of TFA maybe regulate by the expression of KDR also. But it can not inhibit the vascular endothelial cells apoptosis through raising the expression of bcl-2 protein.