| Objective: TO explore the effect of therapentic on ursolic acid in hemangioma and its possible mechanism,which is through studying the effect of ursolic acid on invitro culture mouse henmangioma endothelial cell(EOMA cells)proliferation and apoptosis,at the same time studying ursolic acid whether influence on vascular endothelial growth factor VEGF and endothelial growth factor receptor-2 VEGF-2 expression.With the experiment,hope to Provide a theoretical basis for finding new treatments for hemangiomas.Methods: EOMA strain in vitro cultivation of which group B1 as the negative control group and group B2,group B3 as experimental groups,B4 as Positive control group.B1: add 0.1 mlPBS solution;B2: add 0.1 ml 10 mg/L ursolic acid solution;B3: add 0.1 ml 20 mg/L ursolic acid solution;B4: add 0.1 ml 10 mg/L propranolol solution.After 0 h,24 h,48 h respectively observing,compare each group cells growth and apoptosis those under different concentration of ursolic acid in vitro culture of EOMA cells.Following is the specific methods.HE staining detection cell morphology is changed.Using a determined by MTT method to detect each cell proliferation.Using flow cytometry(Annexin V/PI staining method)was to develop the determination of each cell apoptosis.Using ELISA test method for detection of VEGF and VEGFR concentration changes.All Experimental data is measured by using the statistical software SPSS20.0 analysis processing,data results with mean + /-standard deviation(x ±s)said.ANOVA was used to compare the effect indicators at each time point,Between the two groups were compared using LSD method,Repeated measurement data comparison between groups using repetitive measure analysis of variance,set P < 0.05 was statistically significant.Results: 1.HE dyeing observation of cell morphology: intervention in 0 h,B2 and B3 are compared with control group B1,there is no significant difference.In intervention for 24 hours and 48 hours later,group B2,B3,B4 are compared with negative control group B1,the groups is lower and sparser than control group in cell growth,but settlement growth is not obvious.B3,B4 group are more significant 2.In 0h time,optical density values of group B1,B2,B3,B4 optical density values were: 0.424±0.003,0.422±0.005,0.422±0.008,0.422± 0.008,the cytostatic rate of group B1,B2,B3,B4 were:0.43±0.16%,0.57±0.22%,0.57±0.23%.In 24 h time,optical density values of group B1,B2,B3,B4 optical density values were:0.665±0.013,0.621±0.004,0.560±0.024,0.577±0.031,the cytostatic rate of group B1,B2,B3,B4 were:6.62±0.23%,15.71±0.50%,13.28±0.36%.In 48 h time,optical density values of group B1,B2,B3,B4 optical density values were:0.879±0.017,0.811±0.032,0.718±0.019,0.745±0.039,the cytostatic rate of group B1,B2,B3,B4 were:7.78±0.41%,18.31±0.31%,15.21±0.55%.Statistical analysis shows that: in 0h time differences among the groups was not statistically significant(P> 0.05);But at 24 h and 48 h,differences among the groups was statistically significant(P <0.05),and the optical density values of group B2,B3,B4 were lower than group B1,In addition B3,B4 lower than B2,the difference was statistically significant(P <0.05),and there was no significant difference between B3 and B4(P> 0.05).3.Followings are the results of flow cytometry(Annexin V/PI staining)groups of apoptosis ratio.Intervention in 0h,the apoptosis rate of each group were:9.606±0.155%,9.828±0.185%,9.778±0.264%,9.746±0.168%.Intervention in 24 h,the apoptosis rate of each group were:11.773±0.412%,14.158±0.408%,17.251±1.324%,17.626±0.466%.I n t e r v e n t i o n i n 4 8 h,t h e a p o p t o s i s r a t e o f e a c h g r o u p w e r e : 11.390±1.584%,14.946±1.470%,19.886±1.319%,19.971±2.519%.Statistical analysis shows that:in 0h time differences among the groups was not statistically significant(P> 0.05);But at 24 h and 48 h,differences among the groups was statistically significant(P <0.05),and the apoptosis rate of group B2,B3,B4 were higher than group B1,In addition B3,B4 lower than B2,the difference was statistically significant(P <0.05),and there was no significant difference between B3 and B4(P> 0.05).4.Followings are the results of VEGF and VEGFR-2 tested by ELISA.Intervention in 0 h,the absorbance values of VEGF in each group were:1.963±0.009,1.957±0.011,1.951±0.007,1.941±0.042,the concentration of VEGF in each group were 168.717±0.914,168.096±1.106,167.511±0.741,166.560±4.141.while the absorbance values of VEGFR-2 in each group were:1.510±0.012、1.506±0.014,1.494±0.020,1.518±0.013.the concentration of VEGFR-2 in each group were:2748.299±23.564,2740.891±27.786,2715.728±39.654,2764.186±25.257.Intervention in 24 h,the absorbance values of VEGF in each group were 1.952±0.005,1.666±0.008,1.285±0.013,1.264±0.168,the concentration of VEGF in each group were:167.659±0.460,139.303±0.788,101.583±1.326,99.519±16.672.while the absorbance values of VEGFR-2 in each group were:1.492±0.012,1.183±0.012,0.998±0.017,0.989±0.006,the concentration of VEGFR-2 in each group were:2712.658±24.758,2093.773±24.508,1724.705±33.030,1705.893±11.665.Interv ention in 48 h,the absorbance values of VEGF in each group were 1.956±0.007,1.385±0.009,1.187±0.013,1.239±0.153,the concentration of VEGF in each group were168.031±0.716,111.472±0.918,91.905±1.243,97.006±15.155.while the absorbance values of VEGFR-2 in each group were:1.485±0.020,0.879±0.018,0.707±0.016,0.688±0.020,the concentration of VEGFR-2 in each group were:2697.373±39.639,1486.890±36.745,1142.440±32.981,1104.255±41.955.Statistical analysis shows that: in 0h time differences among the groups was not statistically significant(P>0.05);But at 24 h and 48 h,differences among the groups was statistically significant(P <0.05),and the concentration of VEGF and VEGFR-2 of group B2,B3,B4 were lower than group B1,In addition B3,B4 lower than B2,the difference was statistically significant(P <0.05),and there was no significant difference between B3 and B4(P> 0.05).5.Results indicate that drugs in vitro has the effect on inhibiting EOMA cells growth and promoting its apoptosis,reduce the expression of VEGFand VEGFR-2.and after 48 h the effect is more obvious than after 24 h,the effect on concentration of 20 mg/L ursolic acid is more apparent than 10 mg/L,20 mg / L Ursolic acid has the equivalent effect effect as 10 mg / L propranolol.Conclusion: 1.Ursolic acid in vitro inhibitory has the effect on inhibiting EOMA cells growth and promoting its apoptosis.2.Its mechanism of action of EOMA cells may be related with the cut EOMA cells expression of VEGF and VEGFR-2 levels. |
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