Toxicity and Mechanism of Polygoni Multiflori Radix Alcohol Extract on L02 Cells

Yi-qi BAO; Fang SHEN; Yang-lei LI; Dong-ming CHEN; Hong LU

doi:10.13422/j.cnki.syfjx.20201022

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Toxicity and Mechanism of Polygoni Multiflori Radix Alcohol Extract on L02 Cells

Pharmacology | 更新时间：2021-02-09

- Toxicity and Mechanism of Polygoni Multiflori Radix Alcohol Extract on L02 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 10, Pages: 23-28(2020)
- 作者机构：
  
  浙江中医药大学　药学院，杭州　 311400
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20201022
  CLC： R22; R242; R2-031;R285.5
- Received：18 September 2019，
  
  Published Online：07 February 2020，
  
  Published：20 May 2020
- 稿件说明：
移动端阅览
Yi-qi BAO, Fang SHEN, Yang-lei LI, et al. Toxicity and Mechanism of Polygoni Multiflori Radix Alcohol Extract on L02 Cells[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(10): 23-28.
DOI：

Yi-qi BAO, Fang SHEN, Yang-lei LI, et al. Toxicity and Mechanism of Polygoni Multiflori Radix Alcohol Extract on L02 Cells[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(10): 23-28. DOI： 10.13422/j.cnki.syfjx.20201022.

摘要

目的：

探究何首乌醇提物(PME)对人正常肝细胞L02的毒性损伤和作用机制，为何首乌合理安全用药提供依据。

方法：

以PME(5，10，20 g·L

-1

)作用于L02细胞，噻唑蓝(MTT)比色法检测细胞活性；Hoechst 33342染色法观察细胞核形态；Annexin V-FITC/碘化丙啶(PI)双染法检测细胞凋亡率；相关试剂盒检测细胞中乳酸脱氢酶(LDH)释放率、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量；JC-1法检测细胞线粒体膜电位(MMP)变化；流式细胞术检测活性氧(ROS)水平；蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2(Bcl-2)，Bcl-2相关X蛋白(Bax)，半胱氨酸天冬氨酸蛋白酶-9前体(proCaspase-9)及半胱氨酸天冬氨酸蛋白酶-3前体(proCaspase-3)的表达水平。

结果：

与空白组比较，L02细胞在PME作用下存活率下降，呈时间浓度依赖性；Hoechst 33342染色后荧光下可见细胞核皱缩，碎裂，染色质凝集；Annexin V-FITC/PI双染法结果表明PME 20 g·L

-1

组凋亡率上升；PME 20 g·L

-1

组LDH释放率显著增加(

0.01)，细胞内ROS水平显著上升(

0.01)，SOD活力显著下降(

0.01)，PME 5，10，20 g·L

-1

组MMP明显降低(

0.05)。与空白组比较，随着PME给药组浓度增加，PME 10，20 g·L

-1

组proCaspase-3，proCaspase-9蛋白表达水平均显著降低(

0.01)，PME 5，10，20 g·L

-1

组Bax蛋白表达水平明显升高(

0.01)，PME 20 g·L

-1

组Bcl-2蛋白表达水平明显降低(

0.05)。

结论：

PME对L02细胞存在毒性损伤作用，可一定程度破坏肝细胞的结构，促进ROS水平升高，诱导氧化应激，激活线粒体途径，活化凋亡通路相关蛋白引起肝细胞损伤，提示ROS介导的线粒体通路参与了PME诱导肝细胞凋亡的过程。

Abstract

Objective:

To study the toxic effect of Polygoni Multiflori Radix alcohol extract (PME) on L02 cells and the mechanism of ROS inducing apoptosis via mitochondria pathway

so as to provide a basis for the rational and safe administration of Polygoni Multiflori Radix in clinic.

Method:

The 4

5-dimethly-2-thiazolyl-2

5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect the cell viability of PME at different concentrations (5

20 g·L

-1

). Nuclear morphology was observed by Hoechst 33342 staining. The apoptosis rate of cells was detected by Annexin V-FITC/PI. The release rate of lactate dehydrogenase (LDH)

the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the cells were detected by kit instruction. The changes of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were detected by flow cytometry. The relative protein expression levels of B-cell lymphoma-2 (Bcl-2)

Bcl-2-associated X (Bax)

cysteinyl aspartate proteinase-9(proCaspase-9) and cysteinyl aspartate proteinase-3 (proCaspase-3) in the PME-administered group were detected by Western blot.

Result:

After treatment with PME at the concentration of 5

20 g·L

-1

the survival rate of L02 cells were decreased in a concentration and time-depended manner. After treatment with PME for L02 cells

nucleus shrinkage

fragmentation and chromatin condensation were observed under fluorescence after Hoechst 33342 staining. Annexin V-FITC/PI double staining showed a upward cell apoptosis rate in PME 20 g·L

-1

group. Compared with the normal control group

the release rate of LDH was significantly increased (

0.01)

the intracellular ROS level was significantly increased (

0.01)

and the SOD activity was significantly decreased (

0.01)

while the MMP rate was significantly decreased in PME 5

20 g·L

-1

groups (

0.05). With the increase in the concentration of PME

proCaspase-3

proCaspase-9

Bcl-2 protein showed a significantly downward trend in PME 10

20 g·L

-1

groups (

0.01)

while the expression of Bax protein was significantly up-regulated in PME 20 g·L

-1

group (

0.05).

Conclusion:

The study illustrated that PME have toxic effects on L02 cells

which may destroy the structure of hepatocytes to a certain extent

promote ROS levels

induce oxidative stress

activate the mitochondrial pathway

and then activate apoptosis-related proteins to cause cells damage. It is suggested that ROS-mediated mitochondrial pathway was involved in PME-induced apoptosis.

关键词

Keywords

references

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