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

Xiaoyuan ZHANG; Ziteng MA; Yunfang JIA; Guiqiong HE

doi:10.16016/j.2097-0927.202403052

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

Basic Medicine | 更新时间：2026-01-09

- Knockdown of equilibrative nucleotide transporter 1 protects against Alzheimer's disease by reducing inflammatory response
- Journal of Army Medical University Vol. 46, Issue 23, Pages: 2588-2598(2024)
- 作者机构：
  
  1.400016 重庆，重庆医科大学：神经科学研究中心
  2.400016 重庆，重庆医科大学：基础医学院生理教研室
  3.400016 重庆，重庆医科大学：基础医学院解剖教研室
- 作者简介：
- 基金信息：
  
  the Scientific and Technological Research Project for Young Scholars of Chongqing Education Commission(KJQN202200454)
- DOI：10.16016/j.2097-0927.202403052
  CLC： R394-33;R394.3;R745.7
- Received：12 March 2024，
  
  Revised：2024-04-19，
  
  Published：15 December 2024
- 稿件说明：
移动端阅览
Xiaoyuan ZHANG, Ziteng MA, Yunfang JIA, et al. Knockdown of equilibrative nucleotide transporter 1 protects against Alzheimer's disease by reducing inflammatory response[J]. Journal of Army Medical University, 2024, 46(23): 2588-2598.
DOI：

Xiaoyuan ZHANG, Ziteng MA, Yunfang JIA, et al. Knockdown of equilibrative nucleotide transporter 1 protects against Alzheimer's disease by reducing inflammatory response[J]. Journal of Army Medical University, 2024, 46(23): 2588-2598. DOI： 10.16016/j.2097-0927.202403052.

摘要

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(

0.05)，而当敲低ENT1时细胞存活率显著上调(

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(

0.05)，而敲低ENT1时可逆转N2A-APP细胞内炎症因子水平的上调(

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。

Abstract

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 (

0.05)

while its knockdown increased the cell survival rate (

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 (

0.05)

while ENT1 knockdown reversed the above changes in inflammatory factors in N2A-APP cells (

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.

关键词

Keywords

references

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