Effect of Danggui Shaoyaosan on Copper Ion-mediated Aβ Aggregation in AD Cell Model

Xu-yi HAO; Si LUO; Shu-yi CHENG; Yun-bo CHEN

doi:10.13422/j.cnki.syfjx.20190539

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Effect of Danggui Shaoyaosan on Copper Ion-mediated Aβ Aggregation in AD Cell Model

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- Effect of Danggui Shaoyaosan on Copper Ion-mediated Aβ Aggregation in AD Cell Model
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 6, Pages: 45-51(2019)
- 作者机构：
  
  广州中医药大学　临床药理研究所，广州　 510405
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20190539
  CLC： R289;R742;R246.6
- Received：04 September 2018，
  
  Published Online：12 May 2018，
  
  Published：20 March 2019
- 稿件说明：
移动端阅览
Xu-yi HAO, Si LUO, Shu-yi CHENG, et al. Effect of Danggui Shaoyaosan on Copper Ion-mediated Aβ Aggregation in AD Cell Model[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(6): 45-51.
DOI：

Xu-yi HAO, Si LUO, Shu-yi CHENG, et al. Effect of Danggui Shaoyaosan on Copper Ion-mediated Aβ Aggregation in AD Cell Model[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(6): 45-51. DOI： 10.13422/j.cnki.syfjx.20190539.

摘要

目的：

以

淀粉样蛋白1-42(A

1-42

)和铜离子(Cu

2＋

)络合物作用的人神经母细胞瘤(SH-SY5Y)细胞为阿尔茨海默病(AD)模型，探讨Cu

2＋

对A

聚集方式和神经毒性的影响及当归芍药散对Cu

2＋

介导的A

毒性的AD细胞模型的影响。

方法：

将A

1-42

(20 μmol·L

－1

)与不同浓度硫酸铜(CuSO

，20，40 μmol·L

－1

)反应后用硫黄素T(ThT)染色法检测A

聚集状态，再进行A

1-42

-Cu

2＋

组(20＋20 μmol·L

－1

)，当归芍药散组(1.6，3.2，6.4 mg·L

－1

)ThT染色检测A

聚集状态。培养SH-SY5Y细胞，分别孵育不同浓度的A

1-42

(1.25，2.5，5，10，20，40 μmol·L

－1

)及当归芍药散(1.6，3.2，6.4，12.8 mg·L

－1

)，24 h后行噻唑蓝(MTT)比色法确定A

1-42

损伤浓度和当归芍药散保护浓度。随后将SH-SY5Y细胞分为空白组，A

1-42

-Cu

2＋

组(20＋20 μmol·L

－1

)，当归芍药散组(20 μmol·L

－1

＋20 μmol·L

－1

＋1.6 mg·L

－1

)，空白组加入培养基，共作用24 h后行MTT比色法检测细胞生存率，显微镜拍摄细胞形态，蛋白免疫印迹法(Western blot)检测胞内胞外A

1-42

聚集状况。

结果：

与A

1-42

组比较，Cu

2＋

和A

1-42

结合成更多更大的A

聚集物。与正常组比较，模型组细胞活力明显降低(

0.05，

0.01)，增加细胞内外A

1-42

聚集(

0.05，

0.01)；与模型组比较，当归芍药散能提高A

1-42

-Cu

2＋

损伤的SH-SY5Y的细胞生存率(

0.05，

0.01)，减少胞外A

1-42

蛋白(

0.05，

0.01)。

结论：

2＋

能够增加A

的聚集及毒性；当归芍药散能明显减轻Cu

2＋

介导的A

聚集导致的SH-SY5Y细胞损伤，使A

内吞，减少胞外A

聚集，提高细胞生存率。

Abstract

Objective:

To investigate the effect of copper ion(Cu

) on the aggregation and neurotoxicity of A

and affirm the role of Danggui Shaoyaosan

in vitro

the Neuroblastoma (SH-SY5Y) cells treated with

-amyloid 1-42 (A

1-42

) and Cu

were used as a vitro models of Alzheimer's disease(AD).

Method:

1-42

(20 μmol·L

-1

) was reacted with different concentrations of copper sulfate (CuSO

40 μmol·L

-1

)

and then the thioflavine T (ThT) staining method was used to detect the A

aggregation state. The A

aggregation status was also detected by ThT staining in the A

1-42

-Cu

group(20+ 20 μmol·L

-1

)

and Danggui Shaoyaosan groups(1.6

3.2

6.4 mg·L

-1

). The SH-SY5Y cells were cultured and incubated with different concentrations of A

1-42

(1.25

2.5

40 μmol·L

-1

) and Danggui Shaoyaosan(1.6

3.2

6.4

12.8 mg·L

-1

) for 24 h. Subsequently

SH-SY5Y cells were incubated with A

1-42

(20 μmol·L

-1

) and CuSO

(20 μmol·L

-1

) in the A

1-42

-Cu

group

and incubated with A

1-42

(20 μmol·L

-1

)

Danggui Shaoyaosan (1.6 mg·L

-1

) and CuSO

(20 μmol·L

-1

) in Danggui Shaoyaosan group. Control group was added with the medium. After 24 h of co-action

the cell viability was detected by the methylthiazolyl tetrazolium (MTT) assay. The morphology of the cells was photographed by microscopy. The intracellular extracellular A

1-42

aggregation was detected by Western blot.

Result:

and A

1-42

bound to more and larger A

aggregates compared with the A

1-42

group. Compared with the normal group

cell viability was significantly reduced (

0.05

0.01) and intracellular and extracellular A

1-42

aggregation was increased(

0.05

0.01) in the model group. Danggui Shaoyaosan can increase the cell viability of SH-SY5Y damaged by A

-Cu

(

0.05

0.01)

and reduce A

1-42

protein (

0.05

0.01).

Conclusion:

can increase the aggregation and toxicity of A

; Danggui Shaoyaosan can significantly reduce the damage of SH-SY5Y cells induced by Cu

-mediated A

aggregation

promote A

endocytosis

reduce extracellular A

aggregation and increase cell viability.

关键词

Keywords

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