靶向敲低腺苷转运体1通过降低炎症反应对阿尔茨海默病的保护作用研究

Knockdown of equilibrative nucleotide transporter 1 protects against Alzheimer’s disease by reducing inflammatory response

目的 通过构建腺苷转运体1(equilibrative nucleotide transporter 1,ENT1)过表达和敲低质粒,观察ENT1对阿尔茨海默病(Alzheimer’s disease,AD)的影响及可能的机制。方法 利用分子克隆的方式构建ENT1过表达质粒pAAV-ENT1-mCherry和敲低质粒pAAV-ENT1shRNA-ZsGreen。将过表达质粒转染入小鼠神经元N2A细胞,将敲低质粒转染入含瑞士家族人APP695的N2A细胞(N2A-APP)。结合real-time qPCR和Western blot分别检测ENT1 mRNA和蛋白表达及炎症因子的变化,通过CCK-8试剂盒检测对细胞活性的影响。结果 测序和real-time qPCR验证结果显示小鼠ENT1过表达和敲低质粒已成功构建。CCK-8检测结果显示,过表达ENT1水平24 h内细胞存活率显著下调(P<0.05),而当敲低ENT1时细胞存活率显著上调(P<0.01)。Real-time qPCR检测进一步发现,ENT1过表达可引起细胞内炎症因子IL-1β、TNF-α、C1q-a和C1q-b水平的显著上调(P<0.05),而敲低ENT1时可逆转N2A-APP细胞内炎症因子水平的上调(P<0.05)。结论 靶向敲低ENT1的蛋白水平可通过降低炎症反应从而减轻AD的病理改变,ENT1可能是AD病理机制的一个潜在靶点。

Objective To study the role and mechanisms of equilibrative nucleotide transporter 1(ENT1) on Alzheimer's disease(AD) by constructing ENT1 overexpression and knockdown plasmids.Methods Molecular cloning was used to construct the ENT1 overexpression(pAAV-ENT1-mCherry) and knockdown(pAAV-ENT1shRNA-ZsGreen) plasmids.The overexpression plasmids and the knockdown plasmids were transfected into N2A cells(mouse Neuro A2 cells) and N2A-APP cells(N2A cells stably expressing human APP695).The expression of ENT1 and inflammatory factors at mRNA and protein levels were detected by real-time qPCR and Western blotting,respectively,and the change in cell viability were measured with CCK-8 assay.Results Sequencing and real-time qPCR indicated that ENT1 overexpression and knockdown plasmids were successfully constructed.CCK-8 assay showed that ENT1 overexpression significantly reduced the cell survival rate within 24 h(P<0.05),while its knockdown increased the cell survival rate(P<0.01).Real-time qPCR displayed that overexpression of ENT1 enhanced the expression levels of inflammatory factors,such as IL-1β,TNF-α,C1q-a and C1q-b in N2A cells(P<0.05),while ENT1 knockdown reversed the above changes in inflammatory factors in N2A-APP cells(P<0.05).Conclusion Knockdown of ENT1 attenuates pathological changes in AD by reducing the inflammatory response.ENT1 may be a potential target in the pathological mechanism of AD.