Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis

TAO Heyun; LIU Yuanjie; LI Jiepin; ZENG Shuhong; ZHANG Ying; LIU Shenlin; ZOU Xi

doi:10.13422/j.cnki.syfjx.202202421

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Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis

更新时间：2023-04-28

- Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 11, Pages: 82-88(2023)
- 作者机构：
  
  1.南京中医药大学附属医院，南京 210029
  2.南京中医药大学第一临床医学院，南京 210023
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.202202421
  CLC： R22;R242;R2-031;R285.5
- Published：05 June 2023，
  
  Published Online：04 November 2022，
  
  Received：30 May 2022，
- 稿件说明：
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陶鹤云,刘元杰,李洁玭等.健脾养正消癥汤通过抑制有氧糖酵解对胃癌HGC-27细胞增殖及干细胞特性的影响[J].中国实验方剂学杂志,2023,29(11):82-88.

TAO Heyun,LIU Yuanjie,LI Jiepin,et al.Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(11):82-88.
陶鹤云,刘元杰,李洁玭等.健脾养正消癥汤通过抑制有氧糖酵解对胃癌HGC-27细胞增殖及干细胞特性的影响[J].中国实验方剂学杂志,2023,29(11):82-88. DOI： 10.13422/j.cnki.syfjx.202202421.

TAO Heyun,LIU Yuanjie,LI Jiepin,et al.Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(11):82-88. DOI： 10.13422/j.cnki.syfjx.202202421.

摘要

目的

观察健脾养正消癥汤通过抑制有氧糖酵解过程而对人胃癌细胞HGC-27增殖过程及干细胞特性的影响，并探讨其具体机制。

方法

采用噻唑蓝（MTT）比色法测定健脾养正消癥汤（0.25、0.5、1、2、4、8、16、32 g·L

-1

）处理胃癌细胞HGC-27的存活率及化疗敏感性的变化；细胞克隆形成实验检测健脾养正消癥汤（2、4、8 g·L

-1

）对细胞克隆形成能力的影响；葡萄糖检测试剂盒和乳酸测定试剂盒检测健脾养正消癥汤处理后胃癌细胞有氧糖酵解水平的变化；流式细胞术检测胃癌HGC-27细胞中干细胞亚群的比例；蛋白免疫印迹法（Western blot）检测胃癌细胞中乳酸脱氢酶（LDH）及己糖激酶2（HK2）、葡萄糖转运蛋白1（GLUT1）、丙酮酸激酶同工酶M2（PKM2）等糖酵解相关蛋白的表达及八聚体结合转录因子4（OCT4）、SRY-box转录因子2（SOX2）、Nanog等干细胞特性相关蛋白的表达。

结果

MTT比色法结果表明，与空白组比较，健脾养正消癥汤（0.5、1、2、4、8、16、32 g·L

-1

）组能够抑制胃癌HCG-27细胞活力（

0.05，

0.01），其半数抑制浓度（IC

）为4.83 g·L

-1

，且健脾养正消癥汤能够提高胃癌HGC-27细胞对顺铂化疗的敏感性；克隆形成实验表明，与空白组比较，健脾养正消癥汤（2、4、8 g·L

-1

）均能显著减少胃癌HGC-27细胞克隆形成数（

0.01），且呈现浓度依赖性；流式细胞术表明，与空白组比较，健脾养正消癥汤（2、4、8 g·L

-1

）作用后细胞的CD44

CD24

ALDH

细胞亚群比例率明显降低（

0.05）；葡萄糖检测和乳酸检测表明，与空白组比较，健脾养正消癥汤（2、4、8 g·L

-1

）能有效抑制胃癌细胞的葡萄糖摄取和乳酸生成（

0.01）；Western blot表明，与空白组比较，健脾养正消癥汤（2、4、8 g·L

-1

）能下调SOX2、Nanog、OCT4等干性相关蛋白及PKM2、LDH、GLUT1、HK2等有氧糖酵解途径相关蛋白的水平（

0.05，

0.01），并呈浓度依赖性。

结论

健脾养正消癥汤对胃癌细胞增殖具有明显抑制作用，且能够降低胃癌干细胞特性，其作用机制可能与干预胃癌的糖代谢通路—有氧糖酵解有关。

Abstract

Objective

To observe the effect of Jianpi Yangzheng Xiaozheng decoction （JYXD） on the proliferation and stemness of the human gastric cancer （GC） cell line HGC-27 by inhibiting aerobic glycolysis， and explore the underlying mechanism.

Method

Methyl thiazolyl tetrazolium （MTT） assay was employed to determine the survival rate and chemotherapy sensitivity of HGC-27 cells treated with JYXD （0.25， 0.5， 1， 2， 4， 8， 16， 32 g·L

-1

）. Colony formation assay was employed to detect the effect of JYXD （2， 4， 8 g·L

-1

） on the colony formation of the cells. The aerobic glycolysis level of HGC-27 cells after treatment with JYXD was measured by glucose assay kit and lactic acid assay kit. The proportion of stem cell subsets in HGC-27 cells was detected by flow cytometry. Western blot was employed to determine the expression of glycolysis-associated proteins such as lactate dehydrogenase （LDH）， hexokinase 2 （HK2）， glucose transporter 1 （GLUT1）， and pyruvate kinase isozyme M2 （PKM2）， and the expression of stemness-associated proteins such as octamer-binding transcription factor 4 （OCT4）， SRY-box transcription factor 2 （SOX2）， and Nanog.

Result

JYXD （0.5， 1， 2， 4， 8， 16， 32 g·L

-1

） inhibited the activity of HGC-27 cells （

0.05，

0.01）， with the inhibitory concentration 50（IC

） of 4.83 g·L

-1

， and it improved the sensitivity of HGC-27 cells to cisplatin chemotherapy. Compared with the control group， JYXD （2， 4， 8 g·L

-1

） reduced the colony formation number of HGC-27 cells （

0.01） in a concentration-dependent manner. Flow cytometry showed that compared with that in the control group， the proportion of CD44

CD24

ALDH

population in the cells treated with JYXD （2， 4， 8 g·L

-1

） decreased （

0.05）. In addition， JYXD （2， 4， 8 g·L

-1

） inhibited the glucose uptake and lactic acid production of HGC-27 cells. Western blot showed that compared with the control group， JYXD （2， 4， 8 g·L

-1

） down-regulated the expression levels of SOX2， Nanog， OCT4， PKM2， LDH， GLUT1， and HK2 （

0.05，

0.01） in a concentration-dependent manner.

Conclusion

JYXD may inhibit the proliferation and reduce the stemness of HGC-27 cells by regulating the aerobic glycolysis.

关键词

胃癌健脾养正消癥汤有氧糖酵解肿瘤干细胞特性增殖

Keywords

gastric cancerJianpi Yangzheng Xiaozheng decoctionaerobic glycolysisstemness of cancer cellsproliferation

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