Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis

LI Da; SHEN Xiao; WU Quhui; ZHENG Lei; ZHAO Silin; JIN Zhaohui; XIAO Xuefei; FAN Fuyuan

doi:10.13422/j.cnki.syfjx.20222438

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Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis

更新时间：2023-04-28

- Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 11, Pages: 89-96(2023)
- 作者机构：
  
  1.湖南中医药大学第一附属医院，长沙 410007
  2.湖南中医药大学，长沙 410208
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20222438
  CLC： R2-0;R33;R289;R256.1
- Published：05 June 2023，
  
  Published Online：02 December 2022，
  
  Received：06 June 2022，
- 稿件说明：
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李妲,沈枭,吴趋荟等.柴胡含药血清通过Smad3/Rheb轴调节HFL1细胞凋亡和肌成纤维细胞转化[J].中国实验方剂学杂志,2023,29(11):89-96.

LI Da,SHEN Xiao,WU Quhui,et al.Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(11):89-96.
李妲,沈枭,吴趋荟等.柴胡含药血清通过Smad3/Rheb轴调节HFL1细胞凋亡和肌成纤维细胞转化[J].中国实验方剂学杂志,2023,29(11):89-96. DOI： 10.13422/j.cnki.syfjx.20222438.

LI Da,SHEN Xiao,WU Quhui,et al.Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(11):89-96. DOI： 10.13422/j.cnki.syfjx.20222438.

摘要

目的

利用转化生长因子-

（TGF-

）刺激人胚肺成纤维细胞（HFL1）模拟特发性肺纤维化（IPF）的病理过程，探讨柴胡含药血清对IPF的作用和机制。

方法

采用10 μg·L

-1

TGF-

刺激HFL1细胞，给予不同体积分数柴胡含药血清（5%、10%、15%、20%）干预24 h后，利用细胞增殖与活性检测试剂盒-8（CCK-8）检测细胞增殖率。随后细胞分为空白血清组（20%空白血清）、TGF-

组（20%空白血清和10 μg·L

-1

TGF-

）、TGF-

+柴胡含药血清组（5%空白血清、15%含药血清和10 μg·L

-1

TGF-

）、TGF-

+SIS3组（3 μmol·L

-1

SIS3、20%空白血清和10 μg·L

-1

TGF-

）。采用原位末端标记法（TUNEL）检测细胞凋亡率；通过实时荧光定量聚合酶链式反应（Real-time PCR）检测凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）和肌成纤维细胞标志物

-平滑肌肌动蛋白（

-SMA）mRNA表达情况；通过免疫荧光法检测

-SMA、脑Ras同源蛋白（Rheb）、磷酸化（p）-Smad3蛋白表达；蛋白免疫印迹法（Western blot）检测Rheb、p-Smad3、Smad3蛋白表达。

结果

与空白血清组比较，TGF-

组细胞增殖率明显上升（

0.05）。与TGF-

组比较，TGF-

+15%柴胡含药血清组和TGF-

+20%柴胡含药血清组细胞增殖率显著降低（

0.01）。与空白血清组比较，TGF-

组细胞凋亡率显著下降，Bcl-2、

-SMA mRNA表达增加，Bax mRNA表达减少，

-SMA、Rheb蛋白表达增加，Smad3磷酸化水平明显上升（

0.05）；与TGF-

组比较，TGF-

+柴胡含药血清组和TGF-

+SIS3组细胞凋亡率显著上升，Bcl-2、

-SMA mRNA表达减少，Bax mRNA表达增加，

-SMA、Rheb蛋白表达减少，Smad3磷酸化水平下降（

0.05）。

结论

柴胡能够抑制TGF-

诱导的HFL1细胞增殖和肌成纤维细胞转化，促进成纤维细胞凋亡，该作用可能与Smad3/Rheb轴有关。

Abstract

Objective

Transforming growth factor-

（TGF-

） was used to stimulate human fetal lung fibroblast 1 （HFL1） for simulating the pathological process of idiopathic pulmonary fibrosis （IPF） and thereby the effects and mechanism of medicated serum of Bupleuri Radix against IPF were investigated.

Method

TGF-

（10 μg·L

-1

） was employed to stimulate HFL1， and cells were treated with medicated serum of Bupleuri Radix （5%， 10%， 15%， 20%） for 24 h. Then cell proliferation rate was determined with cell counting kit-8 （CCK-8）. Subsequently， cells were classified into the control group （20% blank serum）， TGF-

group （20% blank serum and 10 μg·L

-1

TGF-

）， TGF-

+ medicated serum of Bupleuri Radix group （5% blank serum， 15% medicated serum， and 10 μg·L

-1

TGF-

）， and TGF-

+ SIS3 group （3 μmol·L

-1

SIS3， 20% blank serum， 10 μg·L

-1

TGF-

）. Based on in situ end labeling （TUNEL） staining， the apoptosis rate was examined， and mRNA expression of apoptosis-related proteins B-cell lymphoma 2 （Bcl-2）， Bcl-2 associated X protein （Bax） and myofibroblast marker

-smooth muscle actin （

-SMA） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression of

-SMA， Ras homolog enriched in brain （Rheb）， and phosphorylated （p）-Smad3 was determined by immunofluorescence. Expression of Rheb， p-Smad3， and Smad3 was examined by Western blot.

Result

The cell proliferation rate of TGF-

group increased compared with that of the control group （

0.05）. The cell proliferation rate of TGF+15% medicated serum of Bupleuri Radix group and TGF+20% medicated serum of Bupleuri Radix group decreased compared with that of the TGF-

group （

0.01）. Compared with the control group， TGF-

group showed decrease in apoptosis rate， increase in mRNA expression of Bcl-2 and

-SMA， reduction in Bax mRNA expression， and rise of

-SMA and Rheb protein expression and p-Smad3 level （

0.05）. Compared with TGF-

group， TGF-

+ medicated serum of Bupleuri Radix group and TGF-

+ SIS3 group demonstrated high apoptosis rate， low Bcl-2 and

-SMA mRNA expression， high Bax mRNA expression， and low

-SMA and Rheb protein expression and p-Smad3 level （

0.05）.

Conclusion

Medicated serum of Bupleuri Radix can inhibit TGF-

-induced HFL1 proliferation and fibroblast-myofibroblast transition and promote fibroblast apoptosis by regulating the Smad3/Rheb axis.

关键词

特发性肺纤维化柴胡成纤维细胞Smad3脑Ras同源蛋白成纤维细胞向肌成纤维细胞转化

Keywords

idiopathic pulmonary fibrosisBupleuri RadixfibroblastSmad3RAS homolog enriched in brainfibroblast-myofibroblast transition

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