| Objective: To investigate the effects of QISU SANJIE Decoction(QSD),a Chinese and Yi medicine compound consisting of membranous milkvetch root,tenacious condorvine root or herb,yunnan manyleaf paris rhizome,spreading hedyotis herband and other drugson the proliferation and apoptosis of Hep G2 human hepatocellular carcinoma cells by in vitro experiments,and to determine the effects of QISU SANJIE Decoction on the expression of PI3K/ AKT/m TOR pathway,in order to explore the possible mechanism of QISU SANJIE Decoction for the treatment of hepatocellular carcinoma,in order to provide an experimental basis for the further development and application of QISU SANJIE Decoction as well as to provide more medical options for cancer patients.Methods:1.CCK-8 method and flow cytometry were used to detect proliferation and apoptosis of human hepatocellular carcinoma Hep G2 cells: after cells were harvested in logarithmic growth phase and plated,after 24 h,48h,and 72 h of cell administration,the cells were treated with the control(complete medium group),low-,medium-,and high-dose(4,8 and 16 mg/m L)QSD groups,and 10 μL CCK-8 was added to each well,respectively.After incubating the cells for 1h with protection from light,the OD value at420 nm was measured using an enzyme calibration,and the rate of cell proliferation was calculated.The logarithmic growth phase cells were plated to 6-well plates and then treated with control group,low,medium and high dose groups of QSD for 48 h.At the end of the treatment,Annexin V-FITC and PI reagents were added to double-stained the cells,and the apoptosis rate of Hep G2 cells was detected by flow cytometry.2.RT-PCR and Western Blot were used to detect the effect of Qisu Sanjie Decoction on PI3 K / AKT / m TOR signaling pathway : The cells in logarithmic growth phase were plated and treated with control group,low,medium and high dose groups of QSD for 48 h.At the end of treatment,RNA was extracted by Trizol,and RNA was reversely transcribed into c DNA.The expression of PI3 K,AKT and m TOR m RNA in cells was detected by RT-PCR.The cells in the logarithmic growth phase were passaged to the culture flask.When the cell density reached 60 %,the cells were treated according to the control group and the low,medium and high dose groups of Qisu Sanjie Decoction.At the end of the treatment,Ripa lysate was used to extract the protein.The expression levels of PI3 K,AKT and m TOR and the expression levels of p-PI3 K,p-AKT and p-m TOR were detected.Results : 1.The results of CCK-8 method showed that QSD was found to have significant antitumour activity on Hep G2 human hepatocellular carcinoma cells and was able to effectively inhibit hepatocellular carcinoma cell proliferation,with significant differences between the groups(P<0.05).Inhibition of QSD on Hep G2 human hepatocellular carcinoma cells was found to have drug concentration and time-dependence,i.e.the higher the QSD concentration and the longer the processing time,the lower the activity of the cells.2.QSD was shown to be able to induce apoptosis of Hep G2 human hepatocellular carcinoma cells by using flow cytometry,in which the rate of apoptosis was highest in the high dose group,and there were no significant differences between the low dose group and the control group,and the rate of hepatocellular carcinoma cell apoptosis was positively related to QSD concentration.3.The results of RT-PCR showed that the expression of PI3 K,AKT and m TOR m RNA in human hepatocellular carcinoma Hep G2 cells after treatment with QSD decreased compared with the control group,among which,the inhibition of AKT m RNA by QSD was the most obvious,the expression of individual m RNAs continuously decreased with increasing drug concentration.4.Using a Western Blot assay,we found that the protein expressions of PI3 K,AKT,m TOR,and phospho-PI3 K,AKT,m TOR in Hep G2 human hepatocellular carcinoma cells were all down-regulated to different extents following treatment with QSD,among which the effect of QSD on p-AKT was of particular significance,and the expressions of each protein gradually decreased with the increase of drug concentration.Conclusion:1.QISU SANJIE Decoction diffusion formulas have been shown to have a significant inhibitory effect on the growth of hepatocellular carcinoma cells and can induce apoptosis of hepatocellular carcinoma cells.2.QISU SANJIE Decoction can negatively regulate the m RNA expression of PI3 K,AKT,m TOR as well as the expression levels of PI3 K,AKT,m TOR and phospho-PI3 K,AKT,m TOR proteins in Hep G2 hepatocellular carcinoma cells,thus confirming that QISU SANJIE Decoction can inhibit hepatocellular carcinoma cell proliferation and induce their apoptosis through inhibition of the signaling pathway PI3K/AKT/m TOR. |
