白头翁皂苷B4对肝癌细胞Huh-7及其荷瘤裸鼠肿瘤的抑制作用及机制研究

Inhibitory Effects and Mechanism of Anemoside B4 on Hepatocellular Carcinoma Huh-7 Cells and Tumorbearing Nude Mice

目的:研究白头翁皂苷B4(AB4)对肝癌细胞Huh-7及其荷瘤裸鼠肿瘤的抑制作用及机制。方法:将Huh-7细胞分为AB4给药组、阴性对照组(等体积培养液)、阳性对照组(5-氟尿嘧啶50μmol/L),运用MTT法检测0、3、6、12、25、50、100、200、400、800、1 600μmol/L AB4作用Huh-7细胞12、24、36、48 h的增殖抑制率,计算半数抑制浓度(IC50);克隆形成试验评估50μmol/L AB4作用Huh-7细胞24 h的克隆细胞数;膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶(AnnexinⅤ-FITC/PI)染色检测50μmol/L AB4作用Huh-7细胞24 h的细胞凋亡率;Western blot法检测50μmol/L AB4作用Huh-7细胞24 h后B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(Caspase-3)、活化胱天蛋白酶3(Cleaved Caspase-3)、活化多腺苷二磷酸核糖聚合酶(Cleaved PARP)等凋亡蛋白的表达。将荷瘤裸鼠随机分为阴性对照组(生理盐水)、阳性对照组(5-氟尿嘧啶50 mg/kg)和AB4低、中、高剂量组(25、50、100mg/kg),每组3只,每天腹腔注射相应药物1次,连续19 d,观察裸鼠肿瘤生长情况,计算抑瘤率;苏木精-伊红(HE)染色观察肿瘤细胞形态变化。结果:AB4对Huh-7细胞的增殖抑制率随给药浓度的增加而增加,但50μmol/L后增加不明显,随作用时间的延长而增加,但作用24 h后增加不明显,AB4的IC50为(56.76±1.756)μmol/L。与阴性对照组比较,50μmol/L AB4作用Huh-7细胞24 h的克隆细胞数明显减少、Bcl-2蛋白表达明显减弱(P<0.05),细胞凋亡率和Bax、Caspase-3、Cleaved Caspase-3、Cleaved PARP蛋白表达均明显增加(P<0.05或P<0.01),且与阳性对照组差异无统计学意义。与阴性对照组比较,AB4低、中、高剂量组和阳性对照组荷瘤裸鼠的瘤体积明显减小(P<0.05),肿瘤细胞轮廓逐渐模糊,出现核固缩、核质不清晰、核碎裂现象,其抑瘤率分别为25.93%、39.15%、46.26%、42.83%。结论:AB4对Huh-7细胞及其荷瘤裸鼠均有抑制作用,其机制可能与上调Bax/Bcl-2比例、活化Caspase-3、裂解PARP、诱导细胞发生凋亡有关。

OBJECTIVE:To study the anti-tumor effect of anemoside B4(AB4)on hepatocellular carcinoma Huh-7 and tumor-bearing nude mice and its mechanism.METHODS:Huh-7 cells were divided into AB4 treatment group,negative control group(constant volume of culture medium)and positive control group(5-fluorouracil 50μmol/L).The inhibitory rate of Huh-7 cell proliferation was tested by MTT after treated with 0,3,6,12,25,50,100,200,400,800,1 600μmol/L AB4 for 12,24,36,48 h;IC50 were calculated.The number of clone cells was evaluated by clone formation tests after Huh-7 cells were treated with 50μmol/L AB4 for 24 h.The apoptosis rate of Huh-7 cells was tested by AnnexinⅤ-FITC/PI double staining after treated with 50μmol/L AB4 for 24 h.The expression of apoptotic proteins such as Bcl-2,Bax,Caspase-3,Cleaved Caspase-3 and Cleaved PARP were tested by Western blot after Huh-7 cells were treated with 50μmol/L AB4 for 24 h.Tumor-bearing nude mice were randomly divided into negative control group(normal saline),positive control group(5-fluorouracil 50 mg/kg),AB4 lwo-dose,medium-dose and high-dose groups(25,50,100 mg/kg),with 3 mice in each group.They were given relevant medicine intraperitoneally,once a day,for consecutive 19 d.The growth of tumor was observed,and anti-tumor rate was also calculated.HE staining was used to observe the morphology of tumor cells.RESULTS:The inhibition rate of AB4 on Huh-7 cell proliferation increased with the increase of concentration of AB4,but it did not increase significantly after reaching 50μmol/L;it increased with the increase of time,but it did not increase significantly after 24 h.The IC50 of AB4 was(56.76±1.756)μmol/L.Compared with negative control group,the number of clone cells was decreased significantly after Huh-7 cells were treated with 50μmol/L AB4 for 24 h,while the protein expression of Bcl-2 was decreased significantly(P<0.05);the apoptotic rate,the protein expression of Bax,Caspase-3,Cleaved Caspase-3 and Cleaved PARP were increased significantly(P<0.05 or P<0.01),there was no statistical significance,compared with positive control group.Compared with negative control group,the volume of tumor was decreased significantly in AB4 low-dose,medium-dose and high-dose groups,positive control group(P<0.05);the outline of tumor cells become more and more blurred;there were nuclear pyknosis,unclear nucleoplasm and nuclear fragmentation;its anti-tumor rates were 25.93%,39.15%,46.26%,42.83%,respectively.CONCLUSIONS:AB4 inhibits Huh-7 cells and tumor-bearing mice,the mechanism of which may be associated with up-regulating the proportion of Bax/Bcl-2,activating Caspase-3,cracking PARP and inducing apoptosis.