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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
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| 篇名： | 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 |
| TITLE： | |
| 摘要： | 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50 μmol/L），运用MTT法检测0、3、6、12、25、50、100、200、400、800、 1 600 μmol/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50 μmol/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（Annexin Ⅴ-FITC/PI）染色检测50 μmol/L AB4作用Huh-7细胞24 h的细胞凋亡率；Western blot法检测50 μmol/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、100 mg/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50 μmol/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，50 μmol/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。 |
| ABSTRACT： | 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 50 μmol/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， 1 600 μmol/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 50 μmol/L AB4 for 24 h. The apoptosis rate of Huh-7 cells was tested by Annexin Ⅴ-FITC/PI double staining after treated with 50 μmol/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 50 μmol/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 50 μmol/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 50 μmol/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. |
| 期刊： | 2019年第30卷第5期 |
| 作者： | 薛淑一，李明春，苗青，周煜 |
| AUTHORS： | XUE Shuyi，LI Mingchun，MIAO Qing，ZHOU Yu |
| 关键字： | 白头翁皂苷B4；肝癌细胞Huh-7；荷瘤裸鼠；抑制作用；机制 |
| KEYWORDS： | Anemoside B4； Hepatocellular carcinoma Huh-7； Tumor-bearing nude mice； Inhibitory effect； Mechanism |
| 阅读数： | 650 次 |
| 本月下载数： | 11 次 |
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