Serum Containing Ethanol Extract of Akebia Trifoliate (Thunb.) Koidz Regulates Proliferation and Apoptosis of BEL-7404 Cells by Inhibiting Glycolysis

OBJECTIVE To investigate the effects of serum containing ethanol extract of Akebia trifoliate(Thunb.) Koidz (EEATK) on proliferation,apoptosis and glycolysis in BEL-7404 cells,and to explore its possible mechanism of action.METHODS Drug-containing serum of EEAK low (crude drug 3.15 g·kg^-1),medium (crude drug 6.30 g·kg^-1),high (crude drug 12.60 g·kg^-1) dose group and blank control group were prepared to act on cultured BEL-7404 cell in vitro.CCK-8 method was used to detect the effect of EEATK drug-containing serum on inhibitory rate of cells.DAPI fluorescence staining method was used to observe the effect of EEATK drug-containing serum on cell apoptosis,Annexin V-FITC/PI double staining method was performed to analyze the effect of EEATK drug containing serum on apoptotic rate of cells.The glucose assay kit and the lactic acid test kit were used to detect the glucose uptake and the lactic acid production in BEL-7404 cells.Western blotting was used to detect the effect of EEATK drug-containing serum on protein expressions of cleaved caspase-3,cleaved poly ADP-ribose polymerase (cleaved PARP),hexokinase 2(HK2),pyruvate kinase M2(PKM2),lactate dehydrogenase A (LDHA),serine⁃threonine kinase 2(Akt2) and phosphorylated serine⁃threonine kinase (p-Akt2) in BEL-7404 cells.RESULTS EEATK drug-containing serum could significantly inhibit the cell proliferation (P<0.05 or P<0.01),and could disperse the nucleus and form apoptotic bodies in the cells.EEATK drug-containing serum could significantly increase the total apoptotic rates of BEL-7404 cells (P<0.01),accompanied by the decrease of glucose uptake and production of lactic acid (all P<0.01).Western blotting result showed that drug-containing serum of EEATK medium and high dose group could not only significantly up-regulate the expressions of cleaved-caspase-3 and cleaved PARP (P<0.05),but also down-regulate the expressions of HK2,PKM2 and LDHA protein (P<0.05 or P<0.01),and it could inhibit the phosphorylation of Akt2 in BEL-7404 cells (P<0.05 or P<0.01).CONCLUSION EEATK drug-containing serum can inhibit the cell proliferation and promote the apoptosis of BEL-7404 cells by inhibiting aerobic glycolysis.