Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ <italic style="font-style: italic">β</italic>-catenin Pathway

Wen-ting ZHAO; Jia-ling SUN; Bin WEN; Hai-tao SUN; Xue-mei YANG; Wei-cong CHEN; Chun-yu HE; Xiao-dan ZHONG; Guan-xin CHEN; Song-qi HE

doi:10.13422/j.cnki.syfjx.20202124

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Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway

更新时间：2021-12-03

- Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 1, Pages: 38-45(2021)
- 作者机构：
  
  1.南方医科大学中医药学院，广州 510515
  2.解放军南部战区空军医院，广州 510602
  3.东莞市中医院，广东东莞 523000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20202124
  CLC： R22;R242;R2-031;R285.5
- Received：25 April 2020，
  
  Published Online：20 August 2020，
  
  Published：05 January 2021
- 稿件说明：
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招文婷,孙嘉玲,文彬等.鳖甲煎丸通过Wnt/β-catenin信号通路逆转大鼠肝卵圆细胞EMT的机制[J].中国实验方剂学杂志,2021,27(01):38-45.

ZHAO Wen-ting,SUN Jia-ling,WEN Bin,et al.Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(01):38-45.
招文婷,孙嘉玲,文彬等.鳖甲煎丸通过Wnt/β-catenin信号通路逆转大鼠肝卵圆细胞EMT的机制[J].中国实验方剂学杂志,2021,27(01):38-45. DOI： 10.13422/j.cnki.syfjx.20202124.

ZHAO Wen-ting,SUN Jia-ling,WEN Bin,et al.Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(01):38-45. DOI： 10.13422/j.cnki.syfjx.20202124.

摘要

目的

研究鳖甲煎丸对转化生长因子-

（TGF-

）诱导的大鼠肝卵圆细胞WB-F344上皮间质转化（EMT）的影响，探讨其逆转EMT改变的作用机制。

方法

将WB-F344细胞随机分为空白组，TGF-

模型组（10 μg·L

-1

TGF-

），鳖甲煎丸低剂量组（10 μg·L

-1

TGF-

+0.55 g·kg

-1

鳖甲煎丸），鳖甲煎丸中剂量组（10 μg·L

-1

TGF-

+1.1 g·kg

-1

鳖甲煎丸），鳖甲煎丸高剂量组（10 μg·L

-1

TGF-

+2.2 g·kg

-1

鳖甲煎丸），除空白组外，均采用TGF-

诱导WB-F344细胞构建EMT模型，分别加入鳖甲煎丸低、中、高剂量含药血清处理细胞，采用细胞划痕实验检测WB-F344细胞迁移能力的改变；使用蛋白免疫印迹法（Western blot）检测E-钙黏蛋白（E-cadherin），N-钙黏蛋白（N-cadherin），波形蛋白（Vimentin）表达的变化；使用实时荧光定量PCR（Real-time PCR）检测

-连环蛋白（

-catenin）mRNA表达的改变；使用细胞免疫荧光法检测

-catenin的表达。

结果

与空白组比较，TGF-

诱导WB-F344细胞4 d，WB-F344细胞间隙从紧密逐渐变得松散，细胞形态由鹅卵石状向成纤维样细胞转变，且E-cadherin蛋白表达降低，N-cadherin蛋白表达升高（

0.01），说明成功构建了WB-F344细胞EMT模型。划痕实验结果显示，与空白组比较，TGF-

模型组WB-F344细胞的迁移能力增强（

0.01）；与TGF-

模型组比较，鳖甲煎丸中、高剂量组可以明显抑制WB-F344细胞的迁移能力（

0.05）。Western blot结果显示，与空白组比较，TGF-

模型组E-cadherin蛋白表达水平降低，N-cadherin，Vimentin蛋白表达水平升高（

0.01）；与TGF-

模型组比较，鳖甲煎丸中、高剂量组E-cadherin蛋白表达升高，N-cadherin，Vimentin蛋白表达降低（

0.05，

0.01）。Real-time PCR结果显示，与空白组比较，TGF-

模型组中

-catenin mRNA表达升高（

0.01）；与TGF-

模型组比较，鳖甲煎丸低、中、高剂量组中

-catenin mRNA的表达降低（

0.01）。细胞免疫荧光结果显示，与空白组比较，TGF-

模型组

-catenin在细胞核内荧光表达增强；与TGF-

模型组比较，鳖甲煎丸低、中、高剂量组

-catenin在细胞核内荧光表达减弱，且鳖甲煎丸对细胞核内

-catenin抑制作用与其浓度呈正相关。

结论

鳖甲煎丸可能通过抑制Wnt/

-catenin信号通路，逆转TGF-

诱导WB-F344细胞的EMT进程，抑制WB-F344细胞的迁移能力。

Abstract

Objective

To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition （EMT） of rat hepatic oval cells induced by transforming growth factor-

（TGF-

）， in order to explore its mechanism in reversing EMT.

Method

WB-F344 cells were divided into five groups： blank group， TGF-

model group （10 μg·L

-1

TGF-

）， low-dose group （10 μg·L

-1

TGF-

+0.55 g·kg

-1

Biejiajian Wan）， medium-dose group （10 μg·L

-1

TGF-

+1.1 g·kg

-1

Biejiajian Wan）， high-dose group （10 μg·L

-1

TGF-

+2.2 g·kg

-1

Biejiajian Wan）. Except blank group， TGF-

was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low， medium and high doses of Biejiajian Wan serum， the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of E-cadherin， N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of

-catenin mRNA. The expression of

-catenin was detected by cell immunofluorescence assay.

Result

Compared with normal WB-F344 cells， the intercellular space of WB-F344 cells became loose from tight， and the morphology changed from cobblestone to fibroblast after TGF-

induced WB-F344 cells for 4 days， and the expression of E-cadherin protein decreased， while the expression of N-cadherin protein increased （

0.01）， indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group， the migration ability of WB-F344 cells in TGF-

model group was enhanced （

0.01）， compared with TGF-

model group， Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells； specifically， the low-dose group had no statistical significance， and the medium and high-dose groups had statistical significance （

0.05）. Western blot results showed that compared with the blank group， the expression of E-cadherin decreased， whereas those of N-cadherin and Vimentin increased in the TGF-

model group （

0.01）， compared with TGF-

model group， E-cadherin protein expression was increased in the low， medium and high-dose groups， while the expressions of N-cadherin and Vimentin was decreased； specifically， the low-dose groups had no statistical significance， and the medium and high dose groups had statistical significance （

0.05，

0.01）. Real-time PCR results showed that compared with the blank group， the mRNA expression of

-catenin in the TGF-

model group was increased （

0.05）， whereas compared with TGF-

model group， the mRNA expression of

-catenin in the low， medium and high-dose groups of Biejiajian Wan was reduced （

0.01）. The results of cellular immunofluorescence showed that compared with the blank group， the fluorescence expression of

-catenin in the cell nucleus was enhanced in the TGF-

model group； and compared with the TGF-

model group， the expression of

-catenin in the cell nucleus of the low， medium and high-dose groups of Biejiajian Wan decreased， and the inhibitory effect of Biejiajian Wan on

-catenin in the cell nucleus was positively correlated with its concentration.

Conclusion

Biejiajian Wan may reverse the EMT process that TGF-

induced WB-F344 cells， and inhibit the migration of WB-F344 cells by inhibiting Wnt/

-catenin signaling pathway.

关键词

Keywords

references

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Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway

DOI：10.13422/j.cnki.syfjx.20202124

摘要

Abstract

关键词

Keywords

references