The Research Of How Total Flavone Of Litchi Chinensis Sonn Induce Apoptosis,represses Migration And Invasion Of HepG2 Cells

Objective: To observe the effects of TFL on the proliferation,migration and invasion of Hep G2,and to detect the transcription level and expression of apoptosis-related genes in Hep G2 cells after TFL intervention,to provide the theoretical basis for the treatment of liver cancer with litchi seed.Methods: The effects of TFL and Cisplatin on the proliferation of Hep G2 cells were measured by MTT method,and the half maximal inhibitory concentration(IC50)of TFL and Cisplatin on Hep G2 cells was calculated at24 h and 48 h,the optimal intervention time and does were selected for the follow-up experiment,cells were divided into blank group,positive group,low dose group of TFL,medium dose group of TFL and high dose group of TFL.The effects of TFL on the migration of Hep G2 cells were examined by the wound healing test and Transwell migration test,The effects on invasion were examined by the Transwell invasion test.Western blot was used to detect the expression of Bax,Bcl-2,Caspase-3 and Caspase-8 in Hep G2 cells;The effect of TFL on the transcription level of these proteins in Hep G2 cells was detected by real-time fluorescent quantitative PCR.Results:1.Cytotoxicity of TFL on Hep G2 was detected by MTT assayThe effects of TFL(TFL 80μg/ml,TFL 120μg/ml,TFL 160μg/ml,TFL200μg/ml,TFL 240μg/ml)on the viability of Hep G2 cells were detected by MTT assay.The results showed that the cell viability of each does group was significantly different from that of the blank group(P<0.01)at 24 h,and the cell viability of 160μg/ml,200μg/ml and 240μg/ml groups were all lower than50%;at 48 h,there were significant differences in cell viability among the blank groups and others(P<0.01),the cell viability of 120 μg/ml,160 μg/ml,200 μg/ml,240 μg/ml groups were all lower than 50%.The IC50 of TFL on Hep G2 cells was 136.7±2.40 μg/ml at 24 h and 106.8±1.11 μg/ml at 48 h.The effects of different does of cisplatin(1μg/ml,2μg/ml,4μg/ml,8μg/ml,16μg/ml,32μg/ml,64μg/ml,128μg/ml)on the proliferation of Hep G2 cells were detected by MTT assay.The results showed that at 24 h,the cell viability of all the does groups was significantly different from that of the blank group(P<0.01)except the 1μg/ml and the 2μg/ml group(P<0.01).The cell viability of the 128μg/ml group was less than 50%;At 48 h,the cell viability of each does group was significantly different from that of the blank group(P<0.01).The cell viability of 8μg/ml group,16μg/ml group,32μg/ml group and 64μg/ml group were all lower than 50%.The IC50 of cisplatin on Hep G2 cells was 58.48 ± 2.04μg/ml at 24 h and 5.15 ± 0.56μg/ml at 48 h.According to the results of the experiment,the cells are divided into: blank Group,positive group(Cisplatin 60μg/ml),low-dose group(TFL 70μg/ml),medium-dose group(TFL 140μg/ml),high-dose group(TFL 210μg/ml).2.Wound healing testAfter drug intervention,the migration distance(P<0.01)and cell migration rate in each group were significantly lower than that in the blank group(P<0.01).In the blank group,the migration distance was 70.64±8.74μm and the migration rate was 10.81%±1.75%;in the positive group,the migration distance was 44.55±2.96μm and the migration rate was 6.78%±0.711%;in the low-dose group,the migration distance was 35.44±6.05μm and the migration rate was 5.8% ± 1.37%;The migration distance in TFL medium-dose group was 16.80±9.75μm,and the migration rate was 2.74%±1.58%.In the high-dose group,after 24 hours intervention of TFL,the cells were damaged seriously and the migration distance could not be calculated.3.Transwell migration testAfter drug intervention,the number of membrane perforation in each group was significantly lower than that in the blank group(P<0.01),the number of membrane perforation in the blank group was 244±9,in positive group was 118±9,in TFL low-dose group was 75±3,in TFL medium-dose group was 52±4,in TFL high-dose group was 40±10.4.Transwell invasion testAfter drug intervention,the number of membrane perforation in each group was significantly lower than that in the blank group(P<0.01).The number of membrane perforation in the blank group was 148 ± 16,in the positive group was 85±5,and in the TFL low-dose group was 48±7;in TFL medium-dose group was 28±6,in TFL high-dose group was 21±4.5.Expression of apoptosis-related proteinsThe results showed that: compared with the blank group,TFL significantly increased the expression of Bax protein(P<0.05).The high-dose of TFL could significantly reduce the expression of Bcl-2 protein(P<0.05),and significantly increase the ratio of Bax/Bcl-2(P<0.05).The medium-dose of TFL could significantly increased the expression of Caspase-3 protein and Caspase-8 protein(P<0.05),and the low-dose of TFL significantly increased the expression of Caspase-3 protein(P<0.05).6.Transcription of apoptosis-related genes The results showed that: compared with the blank group,low and medium doses of TFL significantly increased the transcription level of Bax gene(P<0.05).All doses of TFL intervention could significantly reduce the transcription level of Bcl-2 gene in Hep G2 cells(P<0.01).The TFL Medium-dose could significantly increase the transcription levels of Caspase-3 and Caspase-8 genes(P<0.05).The TFL low-dose group could also significantly increased the transcription level of Caspase-3 gene(P<0.05).Conclusion(s):1.TFL can significantly inhibit the proliferation of Hep G2 cells,which may be related to the apoptosis of Hep G2 cells induced by TFL.2.TFL may promote the apoptosis of Hep G2 cells through exogenous and endogenous apoptotic pathways.3.TFL can significantly reduce the migration and invasion of Hep G2 cells,which maybe related to the apoptosis-promoting effect of TFL on Hep G2 cells.