Effect of Tetrandrine on Col-I and FN in TGF-<italic style="font-style: italic">β</italic><sub>1</sub>-induced MRC-5 Cells

Yuan XI; Hai-jing ZHANG; Yun-hang GAO; Hong-ping HOU; Han LI; Zu-guang YE; Guang-ping ZHANG

doi:10.13422/j.cnki.syfjx.20201237

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Effect of Tetrandrine on Col-I and FN in TGF-β₁-induced MRC-5 Cells

Pharmacology | 更新时间：2020-08-11

- Effect of Tetrandrine on Col-I and FN in TGF-β₁-induced MRC-5 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 12, Pages: 94-99(2020)
- 作者机构：
  
  中国中医科学院中药研究所，北京 100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20201237
  CLC： R2-0;R289;R285
- Published Online：20 April 2020，
  
  Published：20 June 2020
- 稿件说明：
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席苑,张海静,高云航等.汉防己甲素对TGF-β₁诱导的MRC-5细胞中Col-I及FN表达的影响[J].中国实验方剂学杂志,2020,26(12):94-99.

XI Yuan,ZHANG Hai-jing,GAO Yun-hang,et al.Effect of Tetrandrine on Col-I and FN in TGF-β₁-induced MRC-5 Cells[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(12):94-99.
席苑,张海静,高云航等.汉防己甲素对TGF-β₁诱导的MRC-5细胞中Col-I及FN表达的影响[J].中国实验方剂学杂志,2020,26(12):94-99. DOI： 10.13422/j.cnki.syfjx.20201237.

XI Yuan,ZHANG Hai-jing,GAO Yun-hang,et al.Effect of Tetrandrine on Col-I and FN in TGF-β₁-induced MRC-5 Cells[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(12):94-99. DOI： 10.13422/j.cnki.syfjx.20201237.

摘要

目的

探讨汉防己甲素（Tet）对人转化生长因子-

（TGF-

）刺激的人胚肺成纤维细胞MRC-5的影响。

方法

不同质量浓度的TGF-

（0，2.5，5，10，20，40 μg·L

-1

）作用于MRC-5细胞，应用细胞增殖与毒性检测法（CCK-8）检测TGF-

对MRC-5细胞的增殖毒性，蛋白免疫印迹法（Western blot）检测MRC-5细胞中

-平滑肌肌动蛋白（

-SMA）及波形蛋白（Vimentin）表达水平的变化，酶联免疫吸附测定法（ELISA）检测MRC-5细胞上清液中I型胶原（Col-I）及纤维黏连蛋白（FN）表达水平的变化，以筛选出TGF-

活化MRC-5细胞的适宜浓度。结合筛选出的TGF-

的浓度条件，与不同浓度的Tet（0，2.5，5，10，20，40 μmol·L

-1

）共同作用于MRC-5细胞，再次应用CCK-8法筛选TGF-

及Tet共培养对MRC-5细胞的安全浓度，Western blot检测MRC-5细胞中

-SMA及Vimentin表达水平的变化，ELISA检测MRC-5细胞上清液中Col-I及FN表达水平的变化。

结果

与空白组比较，给药24 h时，20 μg·L

-1

及40 μg·L

-1

的TGF-

对MRC-5细胞有毒性作用（

0.05），给药48 h时，10，20，40 μg·L

-1

的TGF-

对MRC-5细胞有毒性作用（

0.05）；加入Tet培养24 h时，对MRC-5细胞的半数抑制浓度（IC

）值为14.07 μmol·L

-1

，培养48 h时，IC

值为7.51 μmol·L

-1

；与空白组比较，5 μg·L

-1

的TGF-

刺激MRC-5细胞时，细胞中

-SMA， FN及Col-I的相对含量明显升高（

0.05），Vimentin 相对含量显著升高（

0.01），10 μg·L

-1

组细胞中

-SMA，Vimentin，FN及Col-I的相对含量显著升高（

0.01）；10 μg·L

-1

的TGF-

与不同浓度的Tet共培养，与TGF-

组比较，Tet 5 μmol·L

-1

组，细胞中

-SMA，Vimentin及FN相对含量显著降低（

0.01），Col-I相对含量明显降低（

0.05），Tet 10 μmol·L

-1

组，细胞中

-SMA，Vimentin，FN及Col-I的相对含量均显著降低（

0.01）。

结论

TGF-

能够升高MRC-5细胞中Col-I，FN等细胞外基质的水平，而Tet可以有效抑制这种变化的发生，提示Tet可能对肺纤维化的形成中，成纤维细胞大量分泌细胞外基质的过程有抑制作用。

Abstract

Objective

To investigate the effect of tetrandrine on transforming growth factor-

（TGF-

）stimulated MRC-5 cells.

Method

Different concentrations of TGF-

2.5

40 μg·L

-1

) were applied to MRC-5 cells. Proliferation toxicity of TGF-

to MRC-5 was detected by cell counting kit-8 (CCK-8) method. Detection of alpha smooth muscle actin (

-SMA) and Vimentin's expression levels in MRC-5 by Western blot. Detection of changes of collagen I(Col-I) and fibronectin (FN)'s expression levels in MRC-5 supernatants by enzyme linked immunosorbent assay(ELISA) kit. And the appropriate concentration of TGF-

activated MRC-5 cells was screened. The appropriate concentration of TGF-

and different concentrations of Tet (0

2.5

40 μmol·L

-1

) were applied to MRC-5 cells

and CCK-8 method was used to screen safe concentration again. Western blot was used to detect changes in

-SMA and Vimentin expression levels in MRC-5 cells

and ELISA method to detect changes in Col-I and FN in MRC-5 cell supernatant.

Result

Compared with the blank group

20，40 μg·L

-1

of TGF-

had toxic effects on MRC-5 cells at 24 hours (

0.05)

and 10，20，40 μg·L

-1

of TGF-

had toxic effects on MRC-5 cells at 48 h (

0.05).When Tet is added for 24 h

the half inhibitory concentration (IC

) value was 14.07 μmol·L

-1

and when cultured for 48 h

the IC

value was 7.51 μmol·L

-1

. Compared with the blank group

the relative contents of

-SMA

FN and Col-I in the 5 μg·L

-1

of TGF-

group were obviously increased (

0.05)

and the relative contents of Vimentin were significantly increased (

0.01)

and the relative contents of FN and Col-I

-SMA and Vimentin in 10 μg·L

-1

group were significantly increased (

0.01). 10 μg·L

-1

of TGF-

was co-cultured with Tet at different concentrations. Compared with the TGF-

group

the relative levels of

-SMA

Vimentin and FN in the 5 μmol·L

-1

of Tet group were significantly reduced (

0.01)

and the relative levels of Col-I were obviously reduced (

0.05). In the Tet 10 μmol·L

-1

group

the relative contents of the

-SMA

Vimentin

FN and Col-I were significantly reduced (

0.01).

Conclusion

TGF-

can increase the levels of Col-I

FN and other extracellular matrices in MRC-5 cells

and Tet can effectively inhibit the occurrence of this change. It is suggested that Tet may inhibit secreting extracellular matrix of fibroblasts in the formation of pulmonary fibrosis.

关键词

Keywords

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⁰

Effect of Tetrandrine on Col-I and FN in TGF-β1-induced MRC-5 Cells

DOI：10.13422/j.cnki.syfjx.20201237

摘要

Abstract

关键词

Keywords

references

Effect of Tetrandrine on Col-I and FN in TGF-β₁-induced MRC-5 Cells