Effect of Danggui Shaoyao San Drug-containing Serum on Expression of p-MLCⅡ and MLCⅡ Protein in HSC-T6 Cells Induced by ET-1

Sha-sha JIANG; Yong-fu PAN; Mo YANG; Yun-lai WANG; Dan-dan YIN; Fan XU

doi:10.13422/j.cnki.syfjx.20190226

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Effect of Danggui Shaoyao San Drug-containing Serum on Expression of p-MLCⅡ and MLCⅡ Protein in HSC-T6 Cells Induced by ET-1

Classic Prescriptions | 更新时间：2021-02-09

- Effect of Danggui Shaoyao San Drug-containing Serum on Expression of p-MLCⅡ and MLCⅡ Protein in HSC-T6 Cells Induced by ET-1
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 2, Pages: 14-19(2019)
- 作者机构：
  
  安徽中医药大学　药学院，中药复方安徽省重点实验室，合肥　 230012
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20190226
  CLC： R2-0; R22; R285.5; R289
- Received：26 June 2018，
  
  Published Online：02 November 2018，
  
  Published：20 January 2019
- 稿件说明：
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Sha-sha JIANG, Yong-fu PAN, Mo YANG, et al. Effect of Danggui Shaoyao San Drug-containing Serum on Expression of p-MLCⅡ and MLCⅡ Protein in HSC-T6 Cells Induced by ET-1[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(2): 14-19.
DOI：

Sha-sha JIANG, Yong-fu PAN, Mo YANG, et al. Effect of Danggui Shaoyao San Drug-containing Serum on Expression of p-MLCⅡ and MLCⅡ Protein in HSC-T6 Cells Induced by ET-1[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(2): 14-19. DOI： 10.13422/j.cnki.syfjx.20190226.

摘要

目的：

探究内皮素-1(endothelin-1，ET-1)对大鼠肝星状细胞HSC-T6内磷酸化肌球蛋白轻链Ⅱ(phosphorylated myosin light chainⅡ，p-MLCⅡ)，肌球蛋白轻链Ⅱ(myosin light chainⅡ，MLCⅡ)蛋白表达的影响及当归芍药散(Danggui Shaoyao San)含药血清对其的干预作用。

方法：

将HSC-T6细胞种板后，各组每孔加入DMEM和终体积分数分别为2.5%，5%，10%，15%，20%的空白大鼠血清，采用噻唑蓝(MTT)比色法测定HSC-T6细胞的活力，筛选出适合的大鼠血清浓度范围；将细胞分为空白血清组(5%，10%，15%)和当归芍药散含药血清组(5%，10%，15%)，酶联免疫吸附测定(ELISA)检测基础状态下细胞培养上清液中ET-1的含量；细胞分为空白血清组(10%)，当归芍药散含药血清低、中、高剂量组(5%，10%，15%)，实时荧光定量聚合酶链式反应(Real-time PCR)检测基础状态下细胞培养上清液中ET-1 mRNA的水平；将细胞分为空白血清组(10%)，模型组(10%)，当归芍药散含药血清低、中、高剂量组(5%，10%，15%)，Y-27632抑制剂组(100 μmol·L

－1

)，除空白血清组外，其余各组均加入10 nmol·L

－1

ET-1诱导HSC-T6细胞，蛋白免疫印迹法(Western blot)检测ET-1诱导的HSC-T6细胞中p-MLCⅡ，MLCⅡ蛋白表达。

结果：

选用血清浓度为5%，10%，15%作为含药血清浓度。与空白血清组比较，当归芍药散含药血清组明显降低基础状态下ET-1含量，ET-1 mRNA相对含量(

0.05，

0.01)。与空白血清组比较，模型组细胞内p-MLCⅡ，MLCⅡ蛋白表达水平均显著升高(

0.01)；与模型组比较，当归芍药散含药血清各剂量组及Y-27632抑制剂组均可明显下调p-MLCⅡ，MLCⅡ蛋白表达(

0.05，

0.01)。

结论：

当归芍药散含药血清可能通过下调ET-1的含量，抑制ET-1的自分泌，从而下调p-MLCⅡ，MLCⅡ蛋白表达。

Abstract

Objective:

To investigate the effect of endothelin-1 (ET-1) on the expression of phosphorylated myosin light chain Ⅱ(p-MLCⅡ)and myosin light chain Ⅱ(MLCⅡ)protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San(DSS)drug-containing serum.

Method:

After HSC-T6 cells were seeded

DMEM and blank rat serum with final concentrations of 2.5%

10%

15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium(MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group (5%

10%

15%) and DSS drug-containing serum group (5%

10%

15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%)

DSS drug-containing serum low (5%)

medium (10%) and high dose (15%) groups. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%)

model group (10%)

DSS drug-containing serum low (5%)

medium (10%)

high dose (15%) groups and Y-27632 inhibitor group (100 μmol·L

-1

). Except the blank serum control group

the other groups all received 10 nmol·L

-1

ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡ and MLCⅡ in HSC-T6 cells induced by ET-1.

Result:

Serum concentrations of 5%

10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group

the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state (

0.05

0.01). As compared with the blank serum control group

the expression of p-MLCⅡ and MLCⅡ protein in the model group was significantly increased (

0.01); DSS drug-containing serum groups and Y-27632 inhibitor group can significantly down-regulate p-MLCⅡ and MLCⅡ protein expression (

0.05

0.01).

Conclusion:

DSS drug-containing serum may down-regulate the expression of p-MLCⅡ and MLCⅡ by down-regulating the content of ET-1 and inhibiting the autocrine of ET-1.

关键词

Keywords

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