八月札乙醇提取物含药血清通过抑制糖酵解调控BEL-7404细胞增殖与凋亡

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

关键词:  八月札乙醇提取物  肝癌  增殖  凋亡  有氧糖酵解

Key words:  Akebia trifoliate ethanol extract  hepatic carcinoma  proliferation  apoptosis  aerobic glycolysis

摘要:

目的 研究八月札乙醇提取物含药血清对BEL-7404细胞增殖和凋亡及糖酵解的影响，探讨其可能的作用机制。方法 制备八月札乙醇提取物低(生药3.15 g·kg^–1)、中(生药6.30 g·kg^–1)、高(生药12.60 g·kg^–1)剂量组及空白对照组的含药血清作用于体外培养的BEL-7404细胞。采用CCK-8法检测八月札乙醇提取物含药血清对细胞增殖的影响，采用DAPI荧光染色法观察八月札乙醇提取物含药血清对细胞凋亡的影响，采用Annexin V-FITC/PI双染法检测八月札乙醇提取物含药血清对细胞凋亡率的影响，采用葡萄糖检测试剂盒、乳酸检测试剂盒测定葡萄糖摄取量、乳酸生成量，采用Western blotting检测八月札乙醇提取物含药血清对BEL-7404细胞中活化的半胱氨酸蛋白酶-3(cleaved caspase-3)、活化的多聚腺苷二磷酸核糖聚合酶(cleaved poly ADP-ribose polymerase，cleaved PARP)、己糖激酶2(hexokinase 2，HK2)、丙酮酸激酶(pyruvate kinase M2，PKM2)和乳酸脱氢酶A(lactate dehydrogenase A，LDHA)及丝氨酸/苏氨酸蛋白激酶(serine⁃threonine kinase 2，Akt2)、磷酸化丝氨酸/苏氨酸蛋白激酶(phosphorylated serine⁃threonine kinase，p⁃Akt2)蛋白表达的影响。结果 八月札乙醇提取物含药血清对BEL-7404细胞有明显的增殖抑制作用(P<0.05或P<0.01)，使细胞核分散，细胞内形成凋亡小体。八月札乙醇提取物含药血清能显著升高BEL-7404细胞总凋亡率(P<0.01)，并伴随着葡萄糖摄取量及乳酸生成量显著降低(均P<0.01)，Western blotting检测表明八月札乙醇提取物中、高剂量组含药血清上调了促凋亡蛋白cleaved caspase-3和cleaved PARP的表达(P<0.05)，下调了糖酵解相关酶HK2、PKM2及LDHA的蛋白表达(P<0.05或P<0.01)，抑制Akt2的磷酸化(P<0.05或P<0.01)。结论 八月札乙醇提取物含药血清通过抑制糖酵解从而抑制BEL-7404细胞增殖及促进其凋亡。

Abstract:

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.