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当归芍药散对AD细胞模型铜离子介导的A
β 聚集的影响Effect of Danggui Shaoyaosan on Copper Ion-mediated Aβ Aggregation in AD Cell Model
- 中国实验方剂学杂志 2019年25卷第6期 页码：45-51
- 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：
郝徐艺，在读硕士，从事脑病的临床和实验研究，E-mail：1024515417@qq.com
陈云波，研究员，博士生导师，从事中医药防治脑病的基础研究，E-mail：ybchengz@gzucm.edu.cn
- 基金信息：国家自然科学基金项目(81674040);广东省自然科学基金项目(2014A030313399);广州市科技计划项目(1563000302)
DOI：10.13422/j.cnki.syfjx.20190539
中图分类号： R289;R742;R246.6收稿日期：2018-09-04，
网络出版日期：2018-05-12，
纸质出版日期：2019-03-20
- 稿件说明：
移动端阅览
郝徐艺, 罗思, 程淑意, 等. 当归芍药散对AD细胞模型铜离子介导的A
β 聚集的影响[J]. 中国实验方剂学杂志, 2019,25(6):45-51.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.
郝徐艺, 罗思, 程淑意, 等. 当归芍药散对AD细胞模型铜离子介导的A
β 聚集的影响[J]. 中国实验方剂学杂志, 2019,25(6):45-51. DOI： 10.13422/j.cnki.syfjx.20190539.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：
2
以
β
淀粉样蛋白1-42(A
β
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
β
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
β
毒性的AD细胞模型的影响。
方法：
2
将A
β
1-42
(20 μmol·L
－1
)与不同浓度硫酸铜(CuSO
4
，20，40 μmol·L
－1
)反应后用硫黄素T(ThT)染色法检测A
β
聚集状态，再进行A
β
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
β
聚集状态。培养SH-SY5Y细胞，分别孵育不同浓度的A
β
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
β
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
β
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
β
1-42
聚集状况。
结果：
2
与A
β
1-42
组比较，Cu
2＋
和A
β
1-42
结合成更多更大的A
β
聚集物。与正常组比较，模型组细胞活力明显降低(
P
<
0.05，
P
<
0.01)，增加细胞内外A
β
1-42
聚集(
P
<
0.05，
P
<
0.01)；与模型组比较，当归芍药散能提高A
β
1-42
-Cu
2＋
损伤的SH-SY5Y的细胞生存率(
P
<
0.05，
P
<
0.01)，减少胞外A
β
1-42
蛋白(
P
<
0.05，
P
<
0.01)。
结论：
2
Cu
2＋
能够增加A
β
的聚集及毒性；当归芍药散能明显减轻Cu
2＋
介导的A
β
聚集导致的SH-SY5Y细胞损伤，使A
β
内吞，减少胞外A
β
聚集，提高细胞生存率。
Abstract
Objective:
2
To investigate the effect of copper ion(Cu
2+
) on the aggregation and neurotoxicity of A
β
and affirm the role of Danggui Shaoyaosan
in vitro
the Neuroblastoma (SH-SY5Y) cells treated with
β
-amyloid 1-42 (A
β
1-42
) and Cu
2+
were used as a vitro models of Alzheimer's disease(AD).
Method:
2
A
β
1-42
(20 μmol·L
-1
) was reacted with different concentrations of copper sulfate (CuSO
4
20
40 μmol·L
-1
)
and then the thioflavine T (ThT) staining method was used to detect the A
β
aggregation state. The A
β
aggregation status was also detected by ThT staining in the A
β
1-42
-Cu
2+
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
β
1-42
(1.25
2.5
5
10
20
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
β
1-42
(20 μmol·L
-1
) and CuSO
4
(20 μmol·L
-1
) in the A
β
1-42
-Cu
2+
group
and incubated with A
β
1-42
(20 μmol·L
-1
)
Danggui Shaoyaosan (1.6 mg·L
-1
) and CuSO
4
(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
β
1-42
aggregation was detected by Western blot.
Result:
2
Cu
2+
and A
β
1-42
bound to more and larger A
β
aggregates compared with the A
β
1-42
group. Compared with the normal group
cell viability was significantly reduced (
P
<
0.05
P
<
0.01) and intracellular and extracellular A
β
1-42
aggregation was increased(
P
<
0.05
P
<
0.01) in the model group. Danggui Shaoyaosan can increase the cell viability of SH-SY5Y damaged by A
β
-Cu
2+
(
P
<
0.05
P
<
0.01)
and reduce A
β
1-42
protein (
P
<
0.05
P
<
0.01).
Conclusion:
2
Cu
2+
can increase the aggregation and toxicity of A
β
; Danggui Shaoyaosan can significantly reduce the damage of SH-SY5Y cells induced by Cu
2+
-mediated A
β
aggregation
promote A
β
endocytosis
reduce extracellular A
β
aggregation and increase cell viability.
关键词
Keywords
references
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