[1]吴雅欣,赵锦华,孟清琳,等.YTHDC1介导ABCB6上调诱导AD小鼠神经元细胞铁死亡促进认知功能障碍机制的实验研究[J].现代检验医学杂志,2024,39(06):54-60+95.[doi:10.3969/j.issn.1671-7414.2024.06.009]
　WU Yaxin,ZHAO Jinhua,MENG Qinglin,et al.Experimental Study on the Mechanism of YTHDC1 Mediating Upregulation of ABCB6 and Inducing Neuronal Ferroptosis and Promoting Cognitive Dysfunction in AD Mice[J].Journal of Modern Laboratory Medicine,2024,39(06):54-60+95.[doi:10.3969/j.issn.1671-7414.2024.06.009]
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YTHDC1介导ABCB6上调诱导AD小鼠神经元细胞铁死亡促进认知功能障碍机制的实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年06期
页码:
54-60+95
栏目:
论著
出版日期:
2024-11-15

文章信息/Info

Title:
Experimental Study on the Mechanism of YTHDC1 Mediating Upregulation of ABCB6 and Inducing Neuronal Ferroptosis and Promoting Cognitive Dysfunction in AD Mice
文章编号:
1671-7414（2024）06-054-08
作者:
吴雅欣1; 赵锦华1; 孟清琳2; 潘 娜2; 刘养凤2; 苟英之2
（1. 咸阳市第一人民医院神经内科，陕西咸阳 712000；2. 中国人民解放军空军第九八六医院神经内科，西安 710054）
Author(s):
WU Yaxin1;  ZHAO Jinhua1;  MENG Qinglin2;  PAN Na2;  LIU Yangfeng2;  GOU Yingzhi2
（1．Department of Neurology, the First People’s Hospital of Xianyang, Shaanxi Xianyang 712000, China；2． Department of Neurology, the 986th Hospital of the Chinese People’s Liberation Army Air Forcem, Xi’an 710054， China)
关键词:
阿尔兹海默症; YTH 结构域包含蛋白1; ATP 结合盒B 亚家族转运蛋白6; 铁死亡; 认知功能障碍
分类号:
R-332
DOI:
10.3969/j.issn.1671-7414.2024.06.009
文献标志码:
A
摘要:
目的 研究ATP 结合盒B 亚家族转运蛋白6（ATP-binding cassette subfamily B transporter 6，ABCB6）对阿尔兹海默症（Alzheimer’s disease，AD）小鼠认知功能障碍的影响及其可能的潜在调控分子机制。方法 通过注射β- 淀粉样蛋白（amyloid β-protein，Aβ）构建体内AD 小鼠模型；采用水迷宫测试和Y 迷宫测试评估大鼠学习与记忆能力、空间探索能力。通过神经元HT22 细胞和Aβ 构建体外AD 细胞模型；采用RNA 免疫沉淀（RNA immunoprecipitation，RIP）分析YTH 结构域包含蛋白1（YTH domain containing proteins 1，YTHDC1）与ABCB6 的结合关系；实时定量聚合酶链反应（qRT-PCR）检测过表达和敲低转染效率；Western blot 检测YTHDC1 和ABCB6 蛋白，以及铁死亡相关蛋白[ 溶质载体家族7 成员11（solute carrier family 7 member 11，SLC7A11）、谷胱甘肽过氧化物酶4（glutathioneperoxidase 4，GPX4）] 表达水平；CCK-8检测细胞活力；检测丙二醛（malondialdehyde，MDA）、还原型谷胱甘肽（glutathione，GSH）、活性氧（reactive oxygen species，ROS）水平及Fe2+ 含量。结果 AD 小鼠海马组织及Aβ 诱导的HT22 细胞中ABCB6 mRNA (3.51±0.17 vs 1.02±0.01 , 3.45±0.21 vs 1.02±0.01) 和蛋白(3.25±0.14 vs 1.01±0.01 , 3.14±0.16vs 1.01±0.01) 水平均显著上调，差异具有统计学意义（t=-46.238，-20.349；-50.468，-23.013，均P<0.001）。敲低ABCB6 显著降低AD 小鼠抵达平台的时间、到达平台距离，增加小鼠自发交替率和进入新异臂次数的比值（t=27.007，11.264，24.414，19.901，均P<0.001）。敲低ABCB6 促进HT22 细胞增殖，抑制Aβ 诱导的MDA，Fe2+ 水平上调和GSH 水平下调，减少ROS 生成，促进SLC7A11 和GPX4 蛋白表达（t=2.883 ～ 26.122，均P<0.05）。YTHDC1 蛋白通过与ABCB6 mRNA 结合促进其稳定性，上调ABCB6 蛋白表达。敲低YTHDC1 显著降低ABCB6 蛋白水平（t=18.504，P<0.001），促进HT22 细胞增殖，升高GSH 含量及SLC7A11 和GPX4 蛋白水平，降低MDA 和Fe2+ 含量，抑制ROS 生成（t=4.404 ～ 14.486，均P<0.05）。敲低YTHDC1 可以改善AD 小鼠学习与记忆能力和空间探索能力。过表达ABCB6 可逆转敲低YTHDC1 对HT22 细胞铁死亡和AD 小鼠认知功能障碍的影响。结论 YTHDC1 可能通过介导ABCB6 上调，诱导神经元细胞铁死亡，进而促进AD 小鼠的认知功能障碍发生。
Abstract:
Objective To investigate the effects of ATP-binding cassette subfamily B transporter 6 subfamily B (ABCB6) on cognitive dysfunction in Alzheimer’s disease (AD) rats and its possible potential regulatory molecular mechanisms. Methods Amyloid β-protein (Aβ) was injected to construct the AD mouse model in vivo. Water maze test and Y maze test were used to evaluate the learning and memory ability and space exploration ability of rats. An in vitro AD cell model was constructed by HT22 cells and Aβ. The binding relationship between YTH domain containing 1 (YTHDC1) and ABCB6 was analyzed by RNA immuniprecipitation (RIP). Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect overexpression and knockdown transfection efficiency. Western blot analysis was performed to detect the expression levels of YTHDC1 and ABCB6 proteins, as well as ferroptosis related proteins [Solute Carrier Family 7 Member 11 (SLC7A11), Glutathione peroxidase 4(GPX4)]. Cell viability was detected with CCK-8. Malondialdehyde (MDA), Glutathione (GSH), Reactive oxygen species (ROS) levels and Fe2+ content were analyzed by the assay kit. Results The ABCB6 mRNA (3.51±0.17 vs 1.02±0.01 , 3.45±0.21 vs 1.02±0.01) and protein (3.25±0.14 vs 1.01±0.01 , 3.14±0.16 vs 1.01±0.01) levels in the hippocampus of AD mice and Aβ-induced HT22 cells were up-regulated，and the differences were statistically significant （t=-46.238，-20.349；-50.468，-23.013，all P<0.001）. Knocking down ABCB6 decreased the time and distance of AD mice reaching the platform, and increased the ratio of spontaneous exchange rate to the number of times of entered the new arm (t=27.007，11.264，24.414，19.901，all P<0.001). Knockdown ABCB6 promoted HT22 cell proliferation, decreased levels of MDA and Fe2+, increased GSH levels, reduced ROS generation, and promoted expression of SLC7A11 and GPX4 proteins (t=2.883 ～ 26.122, all P<0.05). YTHDC1 protein promoted its stability by binding to ABCB6 mRNA and up-regulated the expression of ABCB6 protein. Knockdown of YTHDC1 decreased ABCB6 protein level (t=18.504, P<0.001), promoted the proliferation of HT22 cells, increased GSH content, SLC7A11 and GPX4 protein levels, decreased MDA and Fe2+ content, and inhibited ROS production (t=4.404 ～ 14.486, all P<0.05). Knocking down YTHDC1 could improve the learning and memory ability and spatial exploration ability of AD mice. Over-expression of ABCB6 reversed the effects of YTHDC1 knockdown on ferroptosis in HT22 cells and cognitive dysfunction in AD mice. Conclusion YTHDC1 may induce ferroptosis of neuronal cells by mediating the up-regulation of ABCB6, thus promoting cognitive dysfunction in AD mice.

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备注/Memo

备注/Memo:
作者简介： 吴雅欣（1990-），女，硕士，主治医师，研究方向：认知障碍、神经系统免疫疾病、周围神经病，E-mail:wyx6457@163.com。
通讯作者：孟清琳（1979-），男，本科，主治医师，研究方向：脑血管介入，E-mail:MQL0419@163.com。
更新日期/Last Update: 2024-11-15