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

Experimental Study on the Mechanism of YTHDC1 Mediating Upregulation of ABCB6 and Inducing Neuronal Ferroptosis and Promoting Cognitive Dysfunction in AD Mice

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(amyloidβ-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(glutathione peroxidase 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Fe^(2+)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,Fe^(2+)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Fe^(2+)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。

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 Amyloidβ-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 Fe^(2+),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^(2+)proliferation of HT22 cells,increased GSH content,SLC7A11 and GPX4 protein levels,decreased MDA and Fe 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.