Study Of Apoptosis Effects On HepG-2 Induced By Codonopsis Lanceolata Extract

ObjectiveLiver cancer is one of the common malignant tumors in China, highly malignant, growing rapidly. Because of our people greasy food and the increase in hepatitis B patients, morbidity and mortality of liver cancer increase year by year. Therefore, as a major killer of cancer deaths, liver cancer has attracted great attention. Codonopsis lanceolata belongs to diffuse perennial herb, more medicine with its roots, and it is one of folk like eating wild vegetables, used along with type of food and medicine plants. It contains a variety of nutrients and saponins, flavonoids, pentacyclic triterpenoids and other medicinal ingredients, to further explore its role in the prevention and treatment of liver cancer for the foundation. Through culturing the human hepatoma cells HepG-2 in vitro, using the fermentation butanol extract of CL, we investigated the HepG-2 cells on the mechanism of apoptosis., it has opened up new ways for the development of new anti-liver cancer drugs.Methods①We fermented Codonopsis lanceolata with active yeast, and obtained fermented Codonopsis lanceolata extract (FCLE) after ethanol extraction and butanol extraction;②Culturing the HepG-2 cells in vitro, taking lx104 cells, we divided the drug group into 12.5μg/mL、25μg/mL、50μg/mL、100μg/mL、150μg/mL、200μg/mL. The inhibition of HepG-2 cells proliferation was measured by MTT assay, and IC50 of the drug was calculated by SPSS17.0 statistical software;③Culturing the HepG-2 cells in vitro, taking 1x104 cells, using different concentration of FCLE, we observed the HepG-2 morphological characteristics of apoptosis with acridine orange (AO) staining under the fluorescence microscope;④Culturing the HepG-2 cells in vitro, taking 1x104 cells, using different concentration of FCLE, we detected the apoptosis rates by Flow Cytometry;⑤Culturing the HepG-2 cells in vitro, taking 1x104cells, using different concentration of FCLE, we detected the Caspase-3,8,9 activities by Caspase Activity Assay Kits.⑥Culturing the HepG-2 cells in vitro, taking 1x104cells, using different concentration of FCLE, we analyzed the cell cycle by use of the cell cycle and apoptosis detection kits.Results①MTT results showed that, the higher the concentration of drug, the stronger the inhibition for the HepG-2 cell proliferation in a dose-dependent manner, and the IC50 was 239.623μg/mL;②With acridine orange (AO) staining under the fluorescence microscope, the normal cells:the nuclear chromatin structure was normal, the membrane was integrate and the cytoplasmic was in red fluorescence; the apoptotic cells:the nuclear was in yellow-green fluorescence and dense patches or fragmented, showing sudden swelling bubble envelope and apoptotic body;③The results of Flow Cytometry showed that, with the increase of drug concentrations, the rates of apoptosis increased. The cell apoptosis rates of the drug groups(100μg/mL,150μg/mL,200μg/mL) Were higher than the control group significantly(P<0.01), and the cell apoptosis rates among the drug groups were also different significantly (P<0.01). The apoptosis rate of 200μg/mL drug group was significantly higher than 100μg/mL and 150μg/mL drug groups. The apoptosis rate of 150μg/mL drug group was significantly higher than 100μg/mL drug group (P<0.01).④Caspase-3,8,9 activity test results show that, the Caspase-3 activities of the drug groups(100μg/mL,150μg/mL,200μg/mL) were higher than the control group significantly(P<0.01), and the Caspase-3 activities among the drug groups were not different significantly(P>0.05).The Caspase-8 activities of the drug groups(100μg/mL, 150μg/mL,200μg/mL) were higher than the control group significantly (P<0.01), the 200μg/mL group was higher than the 100μg/mL group(P<0.05), and the other drug groups were not different significantly(P>0.05). The Caspase-9 activities of the drug groups(100μg/mL,150μg/mL,200μg/mL)were higher than the control group significantly (P<0.01), the Caspase-9 activities of 150μg/mL、200μg/mL were also higher than 100pμg/mL group significantly(P<0.01), but the 150μg/mL and 200μg/mL had no difference (P>0.05).⑤he test results of cell cycle showed that, the drug groups(100μg/mL,150μg/mL, 200μg/mL) could arrest the cell cycle of HepG-2 cells in G0/G1 phase, and the number of cells of G0/G1 ratio increased significantly, while the S phase and G2/M phase cells reduced significantly. Compared with the control group, the proportion of GO/G1 phase cells for the drug groups(100μg/mL,150μg/mL,200μg/mL) were significantly higher than the control group(P<0.01), but the proportion of cells in S phase were significantly lower than control group(P<0.01).Conclusionsl.The FCLE could inhibit the proliferation of the HepG-2 cells, and with the increase of drug concentrations, the inhibition became stronger;2.The FCLE could promote apoptosis of HepG-2 cells. The higher the drug concentration, the more obvious effects of its pro-apoptotic;3.FCLE made the Caspase-8 activated, downward made the Caspase-9 activated and then the Caspase-3 was activated, finally it induced apoptosis in HepG-2 cells;4. FCLE could arrested HepG-2 cells in G0/G1 phases and had the dose dependent manner.