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烟酰胺单核苷酸腺苷转移酶1对视网膜发育过程中中央碳代谢稳定性的作用
作者:金潇1  秦应祥1  罗凯2 
单位:1. 中国科学院大学重庆仁济医院 眼科, 重庆 400000;
2. 中国科学院大学重庆仁济医院 药剂科, 重庆 400000
关键词:烟酰胺单核苷酸腺苷转移酶1 视网膜 烟酰胺腺嘌呤二核苷酸 中央碳代谢 小鼠 
分类号:R774.1
出版年·卷·期(页码):2023·42·第二期(188-194)
摘要:

目的:探讨烟酰胺单核苷酸腺苷转移酶1(NMNAT1)在视网膜发育过程中对于中央碳代谢的作用。方法:构建NMNAT1条件性敲除小鼠及其同窝对照小鼠模型,使用PCR法进行基因型鉴定。在出生后第4天,取NMNAT1敲除小鼠和同窝对照小鼠的视网膜进行液相色谱串联质谱(LC-MS/MS)靶向代谢组学检测和全转录组测序。代谢物提取物通过Shimadzu LC Nexera X2 UHPLC与QTRAP 5500 LC-MS/MS联用进行分析,并使用 MultiQuant 3.0.3 软件对提取的MRM峰进行积分。在NovaSeq6000平台上利用 Illumina TruSeq Stranded mRNA Library Prep Kit进行全转录组测序,每个样本的总读数深度为100 M。使用FastQC验证读取质量,并使用BBTools 包的Bbduk实用程序修剪引物。使用 HISAT2 2.1.0进行读取比对,使用 StringTie 1.3.6组装和量化转录本。使用DESeq2 R包鉴定差异表达的基因。结果:与对照小鼠相比,NMNAT1敲除小鼠视网膜中共有39种代谢物的含量发生了显著改变(P<0.05);通过代谢物集富集分析发现多种不同生化途径受到了不同程度的破坏,主要包括氨基酸代谢、糖酵解/糖异生、烟酸和烟酰胺代谢以及嘌呤代谢。其中,NMNAT1敲除组小鼠视网膜总NMNAT1水平和烟酸腺嘌呤二核苷酸(NaAD)水平降低约 40%(P<0.05);烟酰胺腺嘌呤二核苷酸磷酸(NADP)和烟酸单核苷酸(NAM)的水平分别降低了约25%和55%(P<0.05);线粒体编码的 NADH 脱氢酶 1(MT-ND1)的水平略有降低(P>0.05);上游糖酵解代谢物葡萄糖、葡萄糖6磷酸(G6P)和1,6-二磷酸果糖(F16bP)的水平大幅升高(P<0.05);磷酸二羟基丙酮(DHAP)和葡萄糖3磷酸(G3P)的水平大幅降低(P<0.05);a-KG和琥珀酸的水平降低了约30%(P<0.05);磷酸肌酸和肌酸的水平略有降低(P>0.5);赤藓糖醇(Erythritol)水平显著降低(P<0.05)。结论:NMNAT1缺失会导致视网膜中严重的特异性代谢缺陷,中央碳、嘌呤核苷酸和氨基酸代谢均受到损害,可能是导致严重视网膜变性的原因。

Objective: To investigate the role of nicotinamide nucleotide adenylyltransferase 1(NMNAT1)in central carbon metabolism during retinal development. Methods: The NMNAT1 conditional knockout mice model and their littermate control mice were constructed, and the PCR method was used for genotype identification. On the fourth day after birth, the retinas of NMNAT1 knockout mice and littermate control mice were taken for liquid chromatography tandem mass spectrometry(LC-MS/MS) targeted metabolomics detection and whole transcriptome sequencing. The metabolite extract was analyzed by Shimadzu LC Nexera X2 UHPLC combined with QTRAP 5500 LC-MS/MS, and the results were analyzed using MultiQuant 3.0.3 software. Whole transcriptome sequencing was performed on the NovaSeq6000 platform using the Illumina TruSeq Stranded mRNA Library Prep Kit, and the total read depth of each sample was 100 M. FastQC was used to verify the read quality, and the Bbduk utility of the BBTools package was used to trim the primers. HISAT2 2.1.0 was used for read comparison, and StringTie 1.3.6 was used to assemble and quantify the transcript. The DESeq2 R package was used to identify differentially expressed genes. Results: Compared with control mice, the content levels of 39 metabolites in the retina of NMNAT1 knockout mice changed significantly(P<0.05). Through metabolite collection enrichment analysis, it was found that many different biochemical pathways were damaged to varying degrees, including amino acid metabolism, glycolysis/gluconeogenesis, niacin and nicotinamide metabolism, and purine metabolism. Among them, for the NMNAT1 knockout group, the total NMNAT1 level and NaAD level were reduced by about 40%(P<0.05); the levels of NADP and NAM were reduced by about 25% and about 55%(P<0.05); the level of NADH was slightly decrease(P>0.05); the levels of upstream glycolytic metabolites glucose, G6P and F16BP are relatively significantly increased(P<0.05); the levels of DHAP and G3P were relatively significantly reduced(P<0.05); the levels of a-KG and succinic acid were reduced by about 30%(P<0.05); the levels of phosphocreatine and creatine were slightly reduced(P>0.5); the level of Erythritol was significantly reduced(P<0.05). Conclusion: Loss of NMNAT1 leads to severe specific metabolic defects in the retina, resulting in impaired central carbon, purine nucleotide, and amino acid metabolism, and may be the cause of severe retinal degeneration.

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