| Objective:In this study, we use Astragalus Membranaceus from Sichuan as raw material and make use of the improved ethyl acetate extraction technology to separate the total Flavonoids of Astragalus(TFA) from Astragalus. Then, we focus our research priorities on the different radioprotective effects of TFA on normal human mesenchymal stem cells (hMSCs) and hepatoma cells injured by irradiation and explore the molecule mechanism of the different radioprotective effects of TFA on normal cells and tumor cells in DNA and protein level.Methods:The HepG-2 hepatocellular carcinoma cells and hMSCs were used as experimental subject. The experiment was set up three experimental groups:blank control group (no irradiation, no TFA), direct irradiation group (radiation only, no TFA), TFA treated irradiation group (TFA treatment+radiation). The experiment cells were irradiated by 60Coγ-ray with a irradiation dose of 6Gy one time when it reached logarithmic growth phase. MTT assay was used to detect the survival rates of hMSCs and HepG-2 cells which were pretreated with different concentrations of TFA before irradiation, and flow cytometer was used to detect the cell apoptosis rates of all the groups in 6h,24h,48h time points. Agarose gel electrophoresis was utilized to examine the DNA ladders of HepG-2 and hMSCs cells which were irradiated by 60Co y-ray before 24 hour or 72 hour. Western blot technique was applied to detect the expression of apoptotic protein on HepG-2 cells such as Fas, Bcl-2 and Bax in all the experiment groups.Results:The MTT results showed that the hMSCs survival rates of TFA treated irradiation group were respectively as 1.15,1.38,1.80 and 1.95 times as the direct irradiation group when the treated concentration of TFA were 0.05mg/ml, 0.10mg/ml,0.15mg/ml and 0.20mg/ml. Inversely, under the same pretreatment, the HepG-2 survival rates of TFA treated irradiation group were respectively as 0.53,0.51,0.41 and 0.23 folds as the direct irradiation group. It indicated that TFA had a strong radioprotection on human normal cells and a significant radiosensitization on tumor cells as well as an evident dose dependent. Agarose gel electrophoresis showed that TFA had different effects on the DNA Ladder of normal cells and tumor cells. Compared with the direct radiation group, the apoptotic DNA ladders of hepatoma cells in TFA treated irradiation group were apparently higher, on the other hand the apoptotic DNA ladders of hMSCs in TFA treated irradiation group were obviously lower. Flow cytometry showed that after being irradiated 6h,24h,48h by 60Coγray the apoptosis rates of hMSCs in the direct radiation group were 29.3%,24.9%,13.6% and the apoptosis rates of hMSCs in the TFA treated irradiation group were 23.3%,11.2%,2.9%; however, the apoptosis rates of HepG-2 in the direct radiation group were 6.9%,9.3%,15.8% and the apoptosis rates of HepG-2 in TFA treated irradiation group were 11.6%,17.3%,20.1%. These results indicate that TFA can decrease the apoptosis in normal cells and promote the apoptosis in tumor cells induced by 60Coγray. It indicated that TFA had a dual role on the regulation of the apoptosis of the cells. The results of Western blot demonstrated that the expression of apoptotic protein Fas on hepatoma cells both in blank control group and in direct irradiation group were poor and similar. However, the expression of Fas in TFA treated irradiation group was higher than that of the direct radiation group and the blank control group significantly. The expressions of Bax in all the groups were the same as Fas. As for the expression of Bcl-2, it was just the opposite expression of Bax and Fas.Conclusion:TFA has obvious effects of radiological protection on human hMSCs, and has not effects of radiological protection and also has effects of apoptosis enhancement on hepatoma cells. TFA can strengthen the lethal effect of 60Coγray on hepatoma carcinoma cell through the expression regulation of apoptosis associated protein.
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