基于DKK3调控探讨藏红花素对Aβ25-35诱导神经元损伤的保护作用  

Neuroprotective Effect of Crocin on Neuron Damage Inducedby Aβ 25-35 by Mediating DKK3

作　　者：杨晓佳[1] 吴敏[1] 江萌[1] 刘立权[1] YANG Xiaojia;WU Min;JIANG Meng;LIU Liquan(Hangzhou Chinese Medicine Hospital,Hangzhou,Zhejian 310007)

机构地区：[1]杭州市中医院,浙江杭州310007

出　　处：《中国中医药科技》2024年第1期27-32,共6页Chinese Journal of Traditional Medical Science and Technology

基　　金：浙江省中医药科技计划项目(2022ZB236)。

摘　　要：目的:探究藏红花素通过调控dickkopf WNT信号通路抑制因子3 (Dickkopf3,DKK3)对Aβ25-35诱导神经细胞损伤的保护作用及机制。方法:Aβ25-35诱导小鼠海马神经细胞系HT22细胞模拟细胞损伤,CCK8增殖实验检测藏红花素最佳干预浓度。qPCR检测DKK3沉默质粒(DKK3 siRNA)以及DKK3过表达质粒(DKK3-OE)的DKK3 mRNA表达,随后进行细胞转染。细胞分为正常组、Aβ25-35组、Aβ25-35+藏红花素组(1.0μmol/L)、Aβ25-35+DKK3 siRNA组、Aβ25-35+siRNA对照组、DKK3-OE组、空白质粒对照组、DKK3-OE+藏红花素组(1.0μmol/L)组。采用流式细胞数检测细胞凋亡,Tubulin β染色观察细胞神经突触形态,Western blot检测细胞Aβ、DKK3、β-catenin、GSK-3β、p-GSK-3β、Caspase-3、Bax、Bcl-2蛋白表达。结果:0.5~2.0μmol/L藏红花素可显著上调HT22细胞活力(P<0.05)。与正常组相比,Aβ25-35组和DKK3-OE组细胞凋亡显著增加,突触长度、分支数显著减少,Aβ、DKK3、p-GSK-3β/GSK-3β、Caspase-3、Bax蛋白表达显著升高,β-catenin、Bcl-2蛋白蛋白表达显著降低(P<0.01)。与DKK3-OE组相比,藏红花素干预则能逆转以上结果。与Aβ25-35组相比,Aβ25-35+藏红花素组和Aβ25-35+DKK3 siRNA组细胞的凋亡显著降低,突触长度、分支数显著增加,Aβ、DKK3、p-GSK-3β/GSK-3β、Caspase-3、Bax蛋白表达显著降低,β-catenin、Bcl-2蛋白蛋白表达显著升高(P<0.05)。结论:藏红花素对Aβ25-35诱导神经细胞损伤具有保护作用,其作用与通过抑制DKK3调控GSK-3β/β-catenin信号通路表达有关。Objective:To study the neuroprotective effect of Crocin mediated Dickkopf3(DKK3)on neuron damage induced by Aβ25-35 and its mechanism.Methods:Mouse hippocampal neuron HT-22 cells were induced by Aβ25-35,then CCK-8 test was used to determine optimal intervention concentration of Crocin.q-PCR was used to detect the DKK3 mRNA expression of DKK3 silencing plasmid(DKK3 siRNA)and DKK3 overexpression plasmid(DKK3-OE),then plasmids were transfected into cells.Cells we divided into normal group,Aβ25-35 group,Aβ25-35+Crocin group(1.0μmol/L),Aβ25-35+DKK3 siRNA group,Aβ25-35+siRNA control group,DKK3-OE group,plasmid control group,DKK3-OE+Crocin group(1.0μmol/L).Flow cytometry assay was used to detect cell apoptosis,staining to observe cell synaptic morphology,Wes-tern blot was used to detect protein expressions of Aβ,DKK3,β-catenin,GSK-3β,p-GSK-3β,Caspase-3,Bax,Bcl-2.Results:0.5~2.0μmol/L Crocin significantly could increase cell activity(P<0.01).Compared to normal group,apoptosis in Aβ25-35 group and DKK3-OE group were significantly increased,synaptic length and number of branches were significantly reduced,and protein expressions of Aβ,DKK3,p-GSK-3β/GSK-3β,Caspase-3 and Bax were significantly increased,the protein expressions ofβ-catenin and Bcl-2 were significantly decreased(P<0.01).Compared to DKK3-OE group,Crocin could reverse the above results.Compared to Aβ25-35 group,Aβ25-35+Crocin group and Aβ25-35+DKK3 siRNA group cell apoptosis were significantly decreased,synaptic length and number of branches were significantly increased,protein expressions of Aβ,DKK3,p-GSK-3β/GSK-3β,Caspase-3 and Bax were significantly decreased,while the protein expressions ofβ-catenin and Bcl-2 were significantly increased(P<0.05).Conclusion:Crocin has a protective effect on injury of nerve cell induced by Aβ25-35,it related to regulate expression of GSK-3β/β-catenin signaling pathway by mediating DKK3.

关 键 词：阿尔兹海默症 藏红花素 DKK3 神经元 GSK-3β/β-catenin信号通路 细胞凋亡 体外实验 

分 类 号：R285[医药卫生—中药学]