Mechanism of Weichang' an on Autophagy of MKN45 Cells

Zhao-yan LI; Wei-xia CHEN; Meng-meng QIN; Jia LI; Yan XU; Ai-guang ZHAO

doi:10.13422/j.cnki.syfjx.20192323

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Mechanism of Weichang' an on Autophagy of MKN45 Cells

Pharmacology | 更新时间：2021-02-09

- Mechanism of Weichang' an on Autophagy of MKN45 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 1, Pages: 71-77(2020)
- 作者机构：
  
  1.上海中医药大学　附属龙华医院，上海　 200032
  2.河南省中医院，郑州　 450002
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20192323
  CLC： R22; R242; R2-031; R285.5
- Received：21 April 2019，
  
  Published Online：19 August 2019，
  
  Published：05 January 2020
- 稿件说明：
移动端阅览
Zhao-yan LI, Wei-xia CHEN, Meng-meng QIN, et al. Mechanism of Weichang' an on Autophagy of MKN45 Cells[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(1): 71-77.
DOI：

Zhao-yan LI, Wei-xia CHEN, Meng-meng QIN, et al. Mechanism of Weichang' an on Autophagy of MKN45 Cells[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(1): 71-77. DOI： 10.13422/j.cnki.syfjx.20192323.

摘要

目的：

观察健脾复方胃肠安对人胃癌MKN45细胞自噬的影响，探讨其抗肿瘤的作用机制。

方法：

体外培养人胃癌MKN45细胞，采用细胞活力检测法(CCK-8)检测不同质量浓度胃肠安(250，500，1 000，2 000 mg·L

-1

)作用体外培养的人胃癌细胞MKN45细胞，共孵育24，48，72 h，检测细胞增殖活力的改变；采用吖啶橙(AO)染色和单丹磺胺戊二胺(MDC)染色观察胃肠安对胃癌MKN45细胞自噬小体及自噬囊泡的变化；实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)分别检测微管相关蛋白1轻链3(LC3)，酵母ATG6同源物(Beclin1)，泛素结合蛋白-1(p62)，自噬相关基因5(ATG5)，自噬相关基因7(ATG7)mRNA和蛋白的表达。

结果：

CCK-8实验显示，与空白组比较，胃肠安(500，1 000，2 000 mg·L

-1

)可明显抑制MKN45细胞的增殖能力(

0.05，

0.01)；AO和MDC染色显示，与空白组比较，随着胃肠安浓度的增加，自噬小体和自噬囊泡含量逐渐增加；Real-time PCR和Western blot结果显示，与空白组比较，胃肠安(1 000 mg·L

-1

)组细胞中自噬相关蛋白LC3-Ⅱ，Beclin1，ATG5，ATG7 mRNA和蛋白表达升高(

0.05)，p62表达下降(

0.05，

0.01)。

结论：

胃肠安可诱导人胃癌MKN45细胞自噬，其机制涉及上调自噬相关基因Beclin1，ATG5，ATG7 mRNA和蛋白表达，下调p62 mRNA和蛋白表达，促进自噬标志蛋白LC3-I向LC3-Ⅱ转化。

Abstract

Objective:

To explore the effect of Weichang' an (WCA) on the autophagy of human gastric cancer MKN45 cells and its possible anti-cancer mechanism.

Method:

MKN45 cells were cultured

in vitro

and incubated with different concentrations (250

500

1 000

2 000 mg·L

-1

) of WCA for 24

72 h. Cell counting kit-8 (CCK-8) assay was used to detect the cell proliferation. AO/EB Dyeing (AO) staining and monodansylcadaverin (MDC) staining were used to observe the changes of the effect of WCA on autophagosome and autophagic vesicles in gastric cancer MKN45 cells at 48 h. Real-time polymerase chain reaction (Real-time PCR) and Western blot were used to detect microtubule-associated protein 1 light chain 3 (LC3)

Beclin1

sequestosome 1 (p62)

human autophagy-related gene 5 (ATG5)

human autophagy-related gene 7 (ATG7) mRNA and protein expression levels.

Result:

WCA showed a dose-and-time-dependent growth inhibition at the concentration above 1 000 mg·L

-1

. Compared with the blank group

WCA (500

1 000

2 000 mg·L

-1

) significantly inhibited the proliferation of MKN45 cells (

0.05

0.01). AO staining and MDC staining showed that autophagosomes and autophagic sacs increased with the rise of WCA concentration compared with the blank group. Real-time PCR and Western blot showed that the expressions of autophagy-related proteins LC3-Ⅱ

Beclin1

ATG5

ATG7

mRNA and protein increased gradually after WCA (1 000 mg·L

-1

) intervention

while p62 expression decreased (

0.05

0.01).

Conclusion:

WCA induces the autophagy of human gastric cancer MKN45 cells in a time-and dose-dependent manner

in vitro

which may be related to the up-regulation of LC3-Ⅱ

Beclin1

ATG5 and ATG7 as well as the down-regulation of p62 and LC3-Ⅰ.

关键词

Keywords

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