Study Of Berberine Combined With Apatinibi Induced Apoptosis In Human Hepatocellular Carcinoma Hep-G2 Cells And Its Mechanism

Objective To study the effect of apatinib,berberine(BBR)and their combination on the proliferation,cycle and apoptosis of hepatocellular carcinoma Hep-G2 cells,and the expression of m RNA and protein of anti-proliferating B cell-related translocation gene-2(BTG2)and cyclin B1 、 Cyclin D1,in order to explore its possible mechanisms;Clarifying whether the drug combination group have a synergistic effect in inhibiting the proliferation of hepatocellular carcinoma cells,promoting cell apoptosis and affecting the m RNA and protein expressions of BTG2,Cyclin B1 and Cyclin D1;And to further explore the effect of Chinese herbal extract BBR in the treatment of liver cancer and feasibility.Methods 1.To investigate the inhibitory effect of Apatinib、berberine on the proliferation of Hep-G2 cells and the time-dose-effect relationship with the methods of CCK8.Taking the one quarter inhibitory concentration of apatinib and berberine Intervention cell for 24 hours as the final concentration of the drug combinationgroup,and further to detect The proliferation inhibition rate of Hep-G2 cells in the drug combination group.2.Flow cytometry detected the cell proliferation and apoptosis of the cells affected by Apatinib group、berberine group and the drug combination group;3.Real-time PCR(RT-PCR)detect the m RNA expression of BTG2 and cyclin B1、Cyclin D1 after being intervened by Apatinib,BBR and the drug combination group.4.Western blotting detect the protein expression of BTG2 and cyclin B1、Cyclin D1 after being intervened by Apatinib,BBR and the drug combination group.Results 1.Effect of Apatinib group,BBR group and group on the proliferation inhibition of Hep-G2 cells.(1)CCK8 detected the cell proliferation inhibition rate of Hep-G2 cells treated with different concentrations of apatinib for 24 h,48h and 72 h.There is a time-dose-dependent relationship between the inhibition rate of Hep-G2 cells and the concentration of Apatinib: with the increase of Apatinib concentration and interventiontime,the inhibitory rate of Hep-G2 cells increased(P<0.01).When the concentration of Apatinib was 0.5μM/L,the Hep-G2 cell inhibitory rate was 25%.(2)CCK8 detected the cell proliferation inhibition rate of Hep-G2 cells treated with different concentrations of Apatinib for 24 h,48h and 72 h.the inhibition rate of Hep-G2 cells was time-dose-dependent with the concentration of BBR.With the increase of BBR concentration and intervention time,the inhibitory rate of Hep-G2 cells increased(P<0.01).When the concentration of BBR was 30μM/L,the Hep-G2 cell inhibitory rate was 25%.(3)According to the results of CCK8,Apatinib(0.5μM/L)and BBR(30 μ M/L)were selected as follow-up experiments and the concentration used in the drug combination group.CCK8 detected the effect of Hep-G2 on the proliferation inhibition of cells affected by Apatinib(0.5μM/L)、 BBR(30μM/L)、the drug combination group(0.5μM/LApatinib+30μM/LBBR)for 24 h,48 h and 72 h.The inhibitory rate of Hep-G2 cells was time-dependent with the Apatinib group and BBR group and the drug combination group.With the extension of the intervention time,the inhibition rate of Hep-G2 cells increased(P<0.01),and the inhibition rate of cell proliferation in the drug combination group was significantly higher than that in the Apatinib group and BBR group(P<0.01).(4)RT-PCR and Western Blot detected the the expression of BTG2 m RNA and protein of Hep-G2 cells treated with Apatinib group,BBR group and the drug combination group for 24 h.Compared with the control group,both Apatinib group,BBR group and the drug combination group could up-regulate the expression of BTG2 m RNA and protein(P<0.01).Compared with Apatinib group and BBR group,the expression of BTG2 m RNA and protein was more significantly up-regulated in the drug combination group(P<0.01),with statistical significance.2.Effects of Apatinib group,BBR group and the drug combination group on cell cycle of Hep-G2 cells.(1)Flow cytometry detected the effect of cell cycle on Hep-G2 cells treated with Apatinib group,BBR group and the drug combination group for 24 h.The results showed that Apatinib group compared with the control group,the percentage of G0/G1 phase significantly increased(P<0.01),and the proportion of cells in S phase and G2/M phase decreased(P<0.01),indicating that Apatinib can block Hep-G2 cells in G0/G1 phase;BBR group compared with the control group,the cell proportion in G2/M phase was significantly increased(P<0.01),and the G0/G1 phase and S phase relatively reduced(P<0.01),indicating that BBR could block Hep-G2 cells in G2/M phase;The drug combination group compared with the control group,the percentage of G0/G1 phase increased significantly(P<0.01),and the cell proportion of S phase and G2/M phase decreased(P<0.01),indicating that the drug combination group could block Hep-G2 cells in G0/G1 phase.(2)RT-PCR and Western Blot detected the the expression of Cyclin B1、Cyclin D1 m RNA and protein of Hep-G2 cells treated with Apatinib group,BBR group and the drug combination group for 24 h.Compared with the control group,both Apatinib group and BBR group and the drug combination group could up-regulate the expression of Cyclin B1、Cyclin D1 m RNA and protein(P<0.01).Compared with Apatinib group and BBR group,the expression of Cyclin B1、Cyclin D1 m RNA and protein was more significantly up-regulated in the drug combination group(P<0.01),and the difference was statistically significant.3.Effects of Apatinib group,BBR group and the drug combination group on cell apoptosis of Hep-G2 cells.Flow cytometry detected the change of cell apoptosis rate of Hep-G2 cells treated with Apatinib group,BBR group and the drug combination group for 24 h,48h and 72 h.The results showed that,compared with the control group,both Apatinib group、BBR group and the drug combination group promote Hep-G2 cell apoptosis;And the apoptosis rate was positively correlated with the intervention time of the three groups(P<0.01).Compared with the two single drug groups at each time point,the apoptosis rate of Hep-G2 cells in the drug combination group was higher(P<0.01 or P<0.05),and the difference was statistically significant.Conclusion 1.Both Apatinib and BBR could inhibit the proliferation of Hep-G2 cells in a dose-and time-dependent manner.The mechanism may be related to up-regulating the expression of anti-proliferative gene BTG2 m RNA and protein;2.The inhibitory effects of Apatinib and BBR on the proliferation of Hep-G2 cells were time-dependent and had additive effects(synergistic inhibition);3.Both Apatinib,BBR and the drug combination group all can induce cell cycle arrest by down-regulating the expression of Cyclin B1、Cyclin D1 m RNA and protein;and can promote cell apoptosis,which is time-dependent;4.From the molecular mechanism level,Apatinib and BBR can provide the theoretical basis for the clinical treatment of liver cancer,and the combination of the two drugs has more obvious effect on cancer.