Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells

Xin-yun DU; Chang-qing LI; Bing CHEN; Xiao-hui LI

doi:10.13422/j.cnki.syfjx.20182422

您当前的位置：

首页 >

文章列表页 >

Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells

Pharmacology | 更新时间：2021-02-09

- Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 2, Pages: 48-54(2019)
- 作者机构：
  
  广州中医药大学　热带医学研究所，中药学院，广州　 510405
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20182422
  CLC： R22; R242; R2-031; R285.5; R273
- Received：05 July 2018，
  
  Published Online：30 September 2018，
  
  Published：20 January 2019
- 稿件说明：
移动端阅览
Xin-yun DU, Chang-qing LI, Bing CHEN, et al. Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(2): 48-54.
DOI：

Xin-yun DU, Chang-qing LI, Bing CHEN, et al. Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(2): 48-54. DOI： 10.13422/j.cnki.syfjx.20182422.

摘要

目的：

观察叶下珠复方Ⅱ号对肝癌 Huh7细胞增殖、凋亡与自噬的影响，进一步探讨其抗肝癌的作用机制。

方法：

体外培养肝癌 Huh7细胞，设空白组，叶下珠复方Ⅱ号高、低质量浓度组(40，20 g·L

－1

)和5-氟尿嘧啶(5-FU)(0.04 g·L

－1

)组，采用噻唑蓝(MTT)比色法检测叶下珠复方Ⅱ号对肝癌 Huh7细胞增殖的影响，流式细胞仪检细胞凋亡变化，单丹磺酰尸胺(MDC)染色观察药物作用后细胞自噬体的变化，实时荧光定量聚合酶链式反应(Real-time PCR)检测各组磷脂酰肌醇3-激酶(PI3K)，丝氨酸-苏氨酸蛋白激酶2(Akt2)，B淋巴细胞瘤-2(Bcl-2)和微管相关蛋白1轻链3(LC3Ⅱ)mRNA表达的改变，蛋白免疫印迹法(Western blot)检测Akt2和LC3Ⅱ蛋白表达的变化。

结果：

叶下珠复方Ⅱ号(40，20 g·L

－1

)可显著抑制肝癌Huh7细胞，并诱导其凋亡，凋亡率分别为51.72%，19.74%，显著高于空白组(

0.01)。采用MDC染色后，叶下珠复方Ⅱ号(40，20 g·L

－1

)组可见大量自噬体的形成。与空白组比较，叶下珠复方Ⅱ号高、低剂量组PI3K，Akt2，Bcl-2 mRNA表达均显著下降，LC3Ⅱ mRNA表达均显著增加(

0.01)。与空白组比较，叶下珠复方Ⅱ号(40，20 g·L

－1

)组处理后的Akt2蛋白表达显著下降，LC3Ⅱ蛋白表达明显增加(

0.05，

0.01)。

结论：

叶下珠复方Ⅱ号对肝癌Huh7细胞增殖有明显的抑制作用，其作用机制可能与抑制PI3K/Akt信号通路活化从而诱导细胞凋亡和自噬有关。

Abstract

Objective:

To observe the effect and investigate the mechanism of compound

Phyllanthus urinaria

Ⅱ(CPU Ⅱ)on proliferation

apoptosis and autophagy of human hepatoma cell line Huh7.

Method:

Huh7 cells were cultured

in vitro

and divided into blank control group

high-dose CPU Ⅱ group (40 g·L

-1

)

low-dose CPU Ⅱ group (20 g·L

-1

)

and 5-FU group (0.04 g·L

-1

). Methye thiazolye telrazlium(MTT) assay was used to detect the inhibitory effect of CPU Ⅱ on proliferation of human hepatoma Huh7 cells. The apoptosis rate was observed by Annexin V-FITC/PI flow cytometry; the changes of autophagosomes in each group were observed by monodansylcadaverin (MDC) staining; Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of phosphatidylinositol 3-kinase (PI3K)

Serine-threonine protein kinase 2(Akt2)

B-cell lympoma-2(Bcl-2) and Microtubule-associated protein 1 light chian 3(LC3Ⅱ) and Western blot was used to detect the protein expression of Akt2 and LC3Ⅱ.

Result:

CPU Ⅱ(40

20 g·L

-1

) significantly inhibited the hepatoma cell line Huh7 and induced apoptosis

with an apoptosis rate of 51.72% and 19.74% respectively

significantly higher than that of control group (

0.01). After MDC staining

it was observed that a large number of fluorescent particles were distributed in the cytoplasm and nucleus of liver cancer cells under the effect of CPU Ⅱ. As compared with blank control group

the mRNA expression levels of PI3K

Akt2

and Bcl-2 were decreased significantly while the mRNA expression of LC3Ⅱ was increased significantly in CPU II high-and low-dose groups (

0.01); the protein expression of Akt was significantly decreased while protein expression of LC3Ⅱwas significantly increased in CPU Ⅱ high-dose and low-dose groups (

0.05

0.01).

Conclusion:

CPUⅡ had obvious inhibitory effect on the proliferation of hepatoma cell line Huh7

and the mechanism may be related to inhibiting the activation of PI3K/Akt signaling pathway to induce apoptosis and autophagy.

关键词

Keywords

references

CHEN W , ZHENG R , Baade P D , et al . Cancer statistics in China，2015 [J]. CA: A Cancer J Clin , 2016 , 66 ( 2 ): 115 - 132 .

葛信艳，黄玮，李瑞琴，等 . PI3K/Akt信号转导通路与肿瘤发生相互作用的机制研究 [J]. 中国现代医药杂志， 2017 , 19 ( 7 ): 98 - 101 .

LI J , ZHOU J , ZHANG D , et al . Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/Akt signalling to reduce the severity of ischaemia /reperfusion-induced lung injury [J]. J Cell Mol Med , 2015 , 19 ( 10 ): 2341 - 2351 .

Singh B N , Kumar D , Shankar S , et al . Rottlerin induces autophagy which leads to apoptotic cell death through inhibition of PI3K/Akt/mTOR pathway in human pancreatic cancer stem cells [J]. Biochem Pharmacol , 2012 , 84 ( 9 ): 1154 - 1163 .

余思云，黄彩梅，胡国华 . 紫草素通过PI3K/Akt信号通路诱导子宫内膜癌Ishikawa细胞凋亡 [J]. 世界中西医结合杂志， 2014 , 9 ( 12 ): 1303 - 1306 .

林琼燕，生秀杰，刘娟，等 . 顺铂通过抑制PI3K/Akt/mTOR信号通路诱导子宫内膜癌Ishikawa细胞自噬 [J]. 肿瘤预防与治疗， 2017 , 30 ( 5 ): 337 - 342 .

Lum J J , Bauer D E , KONG M , et al . Growth factor regulation of autophagy and cell survival in the absence of apoptosis [J]. Cell , 2005 , 120 ( 2 ): 237 - 248 .

查勇，寸英丽，黄云超 . 苦参碱对肝癌细胞株SMMC-7721凋亡和自噬相关基因表达的影响 [J]. 世界华人消化杂志， 2010 , 18 ( 29 ): 3078 - 3083 .

张多强，辛国军，丁洋，等 . 枸杞多糖对HepG2肝癌细胞自噬与细胞凋亡的影响 [J]. 中成药， 2017 , 39 ( 12 ): 2600 - 2602 .

王坤凤，林琛，吴玲芳，等．不同产地叶下珠药材中总酚含量测定 [J]. 中国实验方剂学杂志， 2013 , 19 ( 13 ): 110 - 112 .

罗来育，陈斯泰，李常青，等 . 叶下珠复方Ⅱ号对二乙基亚硝胺诱导大鼠肝癌形成的抑制作用及机制 [J]. 中国实验方剂学杂志， 2016 , 22 ( 8 ): 126 - 132 .

陈滨，郭洁文，何素，等 . 叶下珠复方Ⅱ号对肝癌细胞增殖和胰岛素样生长因子-1受体信号通路转录的作用 [J]. 中国实验方剂学杂志， 2018 , 24 ( 14 ): 108 - 114 .

申建柯，李常青，梁曼仪，等 . 叶下珠复方Ⅱ号对人肝癌HepG2 细胞增殖和细胞凋亡作用 [J]. 中国热带医学， 2011 , 11 ( 7 ): 810 - 812 .

夏曦，赵敏，李常青，等 . 叶下珠复方Ⅱ号对肝星状细胞凋亡和miR-16/Bcl-2表达的影响 [J]. 广州中医药大学学报， 2013 , 30 ( 5 ): 690 - 696,775 .

江南雪，熊墨年，唐晓玲 . 原发性肝癌的中医治疗进展 [J]. 实用中西医结合临床， 2016 , 16 ( 10 ): 84 - 86 .

汤庆丰，孙健，于卉，等 . 基于PI3K/Akt信号通路的中药抗肿瘤多药耐药作用研究进展 [J]. 上海中医药大学学报， 2016 , 30 ( 6 ): 78 - 82 .

王新华，李常青，郭兴伯，等．叶下珠复方联合干扰素治疗慢性乙型肝炎 40 例临床观察 [J]. 中西医结合肝病杂志， 1999 , 8 ( 1 ): 12 - 13 .

Galluzzi L , Pietrocola F , Levine B , et al . Metabolic control of autophagy [J]. Cell , 2014 , 159 ( 6 ): 1263 - 1276 .

Kroemer G , Levine B. Autophagic cell death: the story of a misnomer [J]. Nat Rev Mol Cell Biol , 2008 , 9 ( 12 ): 1004 - 1010 .

Martelli A M , Evangelisti C , Follo M Y , et al . Targeting the phosphatidylinositol 3-kinase /Akt /mammalian target of rapamycin signaling network in cancer stem cells [J]. Curr Med Chem , 2011 , 18 ( 18 ): 2715 - 2726 .

Mizushima N , Yoshimori T , Ohsumi Y. The role of Atg proteins in autophagosome formation [J]. Annu Rev Cell Dev Biol , 2011 , 27 : 107 - 132 .

王树彦，周易明，陈宗祐．自噬与肿瘤的发生和治疗研究进展 [J]. 复旦学报：医学版， 2008 , 35 ( 6 ): 928 - 931,934 .

李婷婷，朱迪，牟童，等 . IL-37通过抑制哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化而诱导SMMC-7721肝癌细胞凋亡和自噬 [J]. 细胞与分子免疫学杂志， 2017 , 33 ( 4 ): 440 - 445 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

No data

Related Author

No data

Related Institution

No data

AI问答

Postal code：100700
Tel：010-84076882 Email：syfjx_2010@188.com
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备11006657号-10 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰