当归芍药散对AD细胞模型铜离子介导的Aβ聚集的影响

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

目的:以β淀粉样蛋白1-42(Aβ1-42)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Aβ聚集方式和神经毒性的影响及当归芍药散对Cu^(2+)介导的Aβ毒性的AD细胞模型的影响。方法:将Aβ1-42(20μmol·L^(-1))与不同浓度硫酸铜(Cu SO4,20,40μmol·L^(-1))反应后用硫黄素T(Th T)染色法检测Aβ聚集状态,再进行Aβ1-42-Cu^(2+)组(20+20μmol·L^(-1)),当归芍药散组(1. 6,3. 2,6. 4 mg·L^(-1)) Th T染色检测Aβ聚集状态。培养SH-SY5Y细胞,分别孵育不同浓度的Aβ1-42(1. 25,2. 5,5,10,20,40μmol·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Aβ1-42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,Aβ1-42-Cu^(2+)组(20+20μmol·L^(-1)),当归芍药散组(20μmol·L^(-1)+20μmol·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Aβ1-42聚集状况。结果:与Aβ1-42组比较,Cu^(2+)和Aβ1-42结合成更多更大的Aβ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Aβ1-42聚集(P <0. 05,P <0. 01);与模型组比较,当归芍药散能提高Aβ1-42-Cu^(2+)损伤的SH-SY5Y的细胞生存率(P <0. 05,P <0. 01),减少胞外Aβ1-42蛋白(P <0. 05,P <0. 01)。结论:Cu^(2+)能够增加Aβ的聚集及毒性;当归芍药散能明显减轻Cu^(2+)介导的Aβ聚集导致的SH-SY5Y细胞损伤,使Aβ内吞,减少胞外Aβ聚集,提高细胞生存率。

Objective: To investigate the effect of copper ion( Cu2+) on the aggregation and neurotoxicity of Aβ,and affirm the role of Danggui Shaoyaosan in vitro,the Neuroblastoma( SH-SY5 Y) cells treated with β-amyloid 1-42( Aβ1-42) and Cu2+were used as a vitro models of Alzheimer’s disease( AD).Method: Aβ1-42( 20 μmol·L-1) was reacted with different concentrations of copper sulfate( CuSO4,20,40 μmol·L-1),and then the thioflavine T( ThT) staining method was used to detect the Aβ aggregation state. The Aβ aggregation status was also detected by ThT staining in the Aβ1-42-Cu2+group( 20 + 20 μmol·L-1),and Danggui Shaoyaosan groups( 1. 6,3. 2,6. 4 mg·L-1). The SH-SY5 Y cells were cultured and incubated with different concentrations of Aβ1-42( 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-SY5 Y cells were incubated with Aβ1-42( 20 μmol·L-1) and CuSO4( 20 μmol·L-1) in the Aβ1-42-Cu2+group,and incubated with Aβ1-42( 20 μmol·L-1),Danggui Shaoyaosan( 1. 6 mg·L-1) and CuSO4( 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 Aβ1-42 aggregation was detected by Western blot. Result: Cu2+and Aβ1-42 bound to more and larger Aβ aggregates compared with the Aβ1-42 group. Compared with the normal group,cell viability was significantly reduced( P < 0. 05,P < 0. 01) and intracellular and extracellular Aβ1-42 aggregation was increased( P < 0. 05, P < 0. 01) in the model group.Danggui Shaoyaosan can increase the cell viability of SH-SY5 Y damaged by Aβ-Cu2+( P < 0. 05,P < 0. 01),and reduce Aβ1-42 protein( P < 0. 05,P < 0. 01). Conclusion: Cu2+can increase the aggregation and toxicity of Aβ;Danggui Shaoyaosan can significantly reduce the damage of SH-SY5 Y cells induced by Cu2+-mediated Aβaggregation,promote Aβ endocytosis,reduce extracellular Aβ aggregation and increase cell viability.