The Research Of Ajuga Ethanol Extract To The Growth Of Mice EOMA Cells

Objective: Adjuvant treatment of traditional Chinese medicine(TCM) is currently a new hotpot in tumor research field. Ajuga is a kind of common traditional TCM, with a long history of application. It is explored to the possible influences on growth of hemangioendothelioma cells of ethanol extract of ajuga and the mechanism is also discussed. It is provided to the cytology foundation and new method for the treatment of hemangioma by ajuga.Methods: Mice hemangioendothelioma(EOMA) cell lines were adopted for in vitro cell culture. Ethanol extract of ajuga was used to intervene the cultured cells. Growth of EOMA cells treated by agents was observed. The proliferation, apoptosis, VEGFR and VEGF expression of cells were detected.Ethanol extract of ajuga obtained by ultrasonic atomization was used as experimental drug. Experimental cells can be divided into negative control group(group B1), which was treated by simple PBS solution, and intervention groups(group B2: 100 μg/m L; group B3: 1mg/m L), which were treated by different concentrations of ethanol extract of ajuga. EOMA cells cultured in vitro were observed for their growth at intervention, 24 h, 48 h and 72 h after intervention. HE staining was used to detect cell morphology changes; Flow cytometry(Annexin V/PI staining) was used to determine apoptosis of 3 groups of cells treated by different concentrations of agents. Ki67 flow cytometry wasused to detect cell proliferation in three groups. RT-PCR was used to detect VEGF m RNA expression. Immunofluorescence staining was used for VEGFR1 expression of each group of cells. Results were expressed as mean ± standard deviation( x ±s). Independent sample t-test was used to compare two mean values. For pairwise comparison of multiple samples, ANOVA was used prior to SNK. SPSS17.0 was used for experimental data. P <0.05 referred to statistically significant differences.Results: After intervention with ethanol extract of ajuga, cell growth was relatively slow and sparse. HE staining showed strip-shaped gathering of a large amount of endothelial cells, with larger nucleus and deeper dyeing compared with treatment groups. 72 h, Ki67 expression in normal control group after the intervention results for160.13±4.57,100μg/m L dosing group was the result of 107.81±11.65,1mg/ m L dosing group was the result of 37.41±1.16. In the first 48 and 72 hour time points after the intervention, 100μg/ml drug group and 1mg/m L drug Ki67 cells mean fluorescence intensity lower than control group, the difference was statistically significant(P<0.05); 1mg/m L drug mean fluorescence intensity values cells compared 100μg/ml low drug group, the difference was statistically significant(P<0.05). After the intervention 72 h, apoptosis in normal control group was the result of 2.15± 0.22, 100μg/m L dosing group was the result of 54.04±4.87,1mg/ m L dosing group was the result of 77.95±2.93. In the first 48 and 72 hour time points after the intervention,100μg/ml drug group,1mg/m L drug group apoptosis rate than the control group, difference wasstatistically significant(P<0.05); 1mg/m L drug group apoptosis rate compared with 100μg/ml drug group, the difference was statistically significant(P<0.05).After the intervention 72 h, 100μg/ml drug group, 1mg/m L VEGF drug group compared with the control group of low density ratio, the difference was statistically significant(P<0.05);The difference of VEGF density ratio between 1mg/m L of drugs and 100μg / ml low drug group was significant statistically(P<0.05).72 h after intervention, immunofluorescence showed VEGFR were all positive expression in each group of cells. There were no obvious differences in VEGFR expression between single cells of different group. But since cell growth was slow and apoptosis was increased in intervention groups, so VEGFR expression within same vision was highest in the control group while lowest in group of 1mg/ml.Conclusion: Ethanol extract of ajuga can inhibit EOMA cells proliferation and growth,and promote its apoptosis in vitro. Its mechanism for EOMA cells may be related to down-regulation of VEGF expression of EOMA cells.