Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling

Xiao-yi FENG; Ning-yi WEI; Wei ZHAO; Wei WANG

doi:10.13422/j.cnki.syfjx.20202121

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Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling

更新时间：2021-12-03

- Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 2, Pages: 52-59(2021)
- 作者机构：
  
  云南中医药大学基础医学院，昆明 650500
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20202121
  CLC： R22;R242;R2-031;R285.5
- Received：14 February 2020，
  
  Published Online：20 August 2020，
  
  Published：20 January 2021
- 稿件说明：
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冯晓异,魏宁颐,赵微等.三七总皂苷通过ERK/Akt通路改变HepG2/阿霉素细胞耐药性[J].中国实验方剂学杂志,2021,27(02):52-59.

FENG Xiao-yi,WEI Ning-yi,ZHAO Wei,et al.Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(02):52-59.
冯晓异,魏宁颐,赵微等.三七总皂苷通过ERK/Akt通路改变HepG2/阿霉素细胞耐药性[J].中国实验方剂学杂志,2021,27(02):52-59. DOI： 10.13422/j.cnki.syfjx.20202121.

FENG Xiao-yi,WEI Ning-yi,ZHAO Wei,et al.Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(02):52-59. DOI： 10.13422/j.cnki.syfjx.20202121.

摘要

目的

观察三七总皂苷对人肝癌阿霉素耐药细胞（HepG2/Adr）耐药性及细胞外调节蛋白激酶（ERK）/蛋白激酶B（Akt）通路主要蛋白表达的影响，从ERK/Akt通路探讨三七总皂苷逆转HepG2/Adr细胞耐药的可能作用机制。

方法

利用噻唑蓝（MTT）比色法检测三七总皂苷对HepG2/Adr细胞增殖的影响，利用（100，50，25 mg·L

-1

）三七总皂苷分别与（20 μmol·L

-1

）阿霉素联合处理HepG2/Adr细胞，同时设置空白组，采用高内涵筛选平台检测药物作用40 min，3 h，6 h阿霉素在HepG2/Adr细胞内的积累；采用蛋白免疫印迹法检测P-糖蛋白/多药耐药基因1/ATP家族所介导的ABC家族转运蛋白（P-gp/MDR1/ABCB1）等耐药相关蛋白及ERK/Akt信号通路主要蛋白表达量；激光共聚焦显微镜检测MDR1在细胞膜上分布的改变。

结果

与人肝癌细胞HepG2比较，HepG2/Adr细胞MDR1表达量显著升高（

0.01）；与阿霉素组比较，三七总皂苷（25，50，100 mg·L

-1

）与阿霉素（20 μmol·L

-1

）联合给药在体外对HepG2/Adr细胞的半数抑制浓度（IC

）显著降低（

0.01），逆转倍数最高为10倍；与阿霉素组比较，联合给药组3，6 h可明显提高阿霉素在细胞内的积累（

0.05），呈剂量依赖性；与空白组和阿霉素组比较，三七总皂苷（50，100 mg·L

-1

）与阿霉素（20 μmol·L

-1

）联合给药组MDR1，ABC半转运蛋白（ABCG2），多药耐药相关蛋白1（MRP1），ERK，磷酸化细胞外调节蛋白激酶（p-ERK），Akt，磷酸化蛋白激酶B（p-Akt），哺乳动物雷帕霉素靶蛋白（mTOR），磷酸化mTOR（p-mTOR）的表达明显降低（

0.05）；与空白组比较，阿霉素组与三七总皂苷（25 mg·L

-1

）组MDR1在细胞膜上的分布差异无统计学意义；与空白组和阿霉素组比较，三七总皂苷（100，50 mg·L

-1

）组可明显减少MDR1在细胞膜上的分布（

0.05）。

结论

三七总皂苷可抑制ERK/Akt通路，降低MDR1的表达，明显提高阿霉素在HepG2/Adr细胞中的积累，可能是三七总皂苷逆转耐药的机制之一。

Abstract

Objective

To investigate the effect of notoginseng total saponins （TNS） on adriamycin （Adr） resistance in HepG2/Adr cells and the expression and activity of the mechanisms as the modulators of multi-drug resistance， so as to explore the possible mechanism of extracellular signal-regulated kinase （ERK） and protein kinase B （Akt） signaling pathways in reversing the resistance of HepG2/Adr cells mechanism.

Method

Effect of TNS on HepG2/Adr cell proliferation was detected by thiazole blue （MTT） method. HepG2/Adr cells were treated with different concentrations （100， 50， 25， 0 mg·L

-1

） of TNS and （20 μmol·L

-1

） Adr respectively， and a blank group was set. The high-content screening platform was used to detect the accumulation of Adr in HepG2/Adr cells after 40 minutes， 3 hours and 6 hours. Western blot was used to detect the expression of P-glycoprotein /multidrug resistance/ATP binding cassette subfamily B member 1（P-gp/MDR1/ABCB1） and other drug resistance-related proteins and the main protein expression of ERK/Akt signaling pathway. The change of MDR1 on cell membranes was observed by laser confocal microscopy.

Result

Compared with HepG2 cells， the expression of MDR1 in HepG2/Adr cells was significantly increased （

0.01）. Compared with the Adr group， the half-inhibitory concentration （IC

） of TNS （25， 50， 100 mg·L

-1

） and Adr （20 μmol·L

-1

） co-administration group on HepG2/Adr cells

in vitro

significantly reduced （

0.01）， and the highest reversal multiple was 10 times. Compared with the Adr group， the co-administration group could significantly increase the accumulation of Adr in the cells （

0.05） in a dose-dependent manner. Compared with the blank group， the co-administration group could significantly reduce MDR1， ABC semitransporter （ABCG2）， multidrug resistance associated protein （MRP1）， ERK， phosphorylated extracellular regulatory protein kinase （p-ERK）， Akt， phosphorylated protein kinase B （p-Akt）， mammals， rapamycin target protein （mTOR） and phosphorylated mammalian rapamycin target protein （p-mTOR）（

0.05）， with the same results in the doxorubicin group. Compared with the blank group， there was no significant difference in the distribution and fluorescence intensity of MDR1 on the cell membrane between the Adr group and the notoginseng total saponins （25 mg·L

-1

） group. Compared with the blank group and the doxorubicin group， TNS could significantly reduce the distribution of MDR1 on the cell membrane （

0.05）.

Conclusion

TNS can inhibit the ERK/Akt pathway， reduce the expression of MDR1， and significantly increase the accumulation of doxorubicin in HepG2/Adr cells， which may be one of the mechanisms of notoginseng total saponins in reversing resistance.

关键词

Keywords

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