Pei Pei, Shen Jinying, He Xuejia, Zeng Yubing, Zhang Ting, Wang Shan
Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
Capital Institute of Pediatrics-Peking University Teaching Hospital, Beijing, China.
Front Cell Dev Biol. 2024 Mar 13;12:1294726. doi: 10.3389/fcell.2024.1294726. eCollection 2024.
In the early stage of embryonic development, the neural tube (NT) cannot be closed properly due to some complex factors, including environmental factors, genetic factors, and the relationship between various factors, leading to the occurrence of neural tube defects (NTDs). In this study, we induced a mouse model of NTDs by feeding mice with a low-folate diet and intraperitoneally injecting them with 1.5 mg/kg methotrexate on E7.5. Fetal mice were achieved at E13.5, and we extracted proteins from brain tissues with trypsin digestion. After enzymatic digestion, peptides were labeled with TMT/iTRAQ and separated in high-performance liquid chromatography (HPLC) for subsequent liquid chromatography tandem mass spectroscopy (LC-MS/MS) analysis. We used gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation to analyze proteomic changes and analyze the functional enrichment of differentially expressed proteins (DEPs) in the NTD mice tissues. A low-folate-induced mouse model was successfully constructed. Folate was used as a sensitizing agent, and the teratogenicity rate of the NTD fetal mice increased to 36.5% when the concentration of methotrexate was at 1.5 mg/kg. Mass spectrometry was used to identify 6,614 proteins, and among them, 5,656 proteins were quantified. In the following proteomic analysis, GO classification and KEGG pathway enrichment analysis were conducted, and heatmaps were drawn for differentially expressed proteins (DEPs). The main pathways associated with NTDs, such as the Hedgehog, Wnt, p53, and Hippo signaling pathways and the one-carbon pool mediated by folate, can be identified through a protein-protein interaction (PPI) network. It was also found that the regulation of ribosomal proteins, such as RPL13 and RPL14, which are upregulated in NTDs, has a certain impact on neural tube development. Our results revealed proteomic changes in the tissues of low-folate-induced NTD mice. Validation showed that ribosomal proteins play a regulatory role during the development of NTDs and provides new ideas for the pathogenesis and preventive measures of NTDs.
在胚胎发育早期，由于一些复杂因素，包括环境因素、遗传因素以及各种因素之间的关系，神经管（NT）无法正常闭合，从而导致神经管缺陷（NTDs）的发生。在本研究中，我们通过给小鼠喂食低叶酸饮食并在胚胎第7.5天腹腔注射1.5mg/kg甲氨蝶呤来诱导建立NTDs小鼠模型。在胚胎第13.5天获得胎鼠，并用胰蛋白酶消化从脑组织中提取蛋白质。酶解后，肽段用TMT/iTRAQ标记，并在高效液相色谱（HPLC）中分离，随后进行液相色谱串联质谱（LC-MS/MS）分析。我们使用基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路注释来分析蛋白质组变化，并分析NTD小鼠组织中差异表达蛋白（DEPs）的功能富集情况。成功构建了低叶酸诱导的小鼠模型。叶酸用作致敏剂，当甲氨蝶呤浓度为1.5mg/kg时，NTD胎鼠的致畸率升至36.5%。使用质谱鉴定了6614种蛋白质，其中5656种蛋白质得到定量。在后续的蛋白质组分析中，进行了GO分类和KEGG通路富集分析，并绘制了差异表达蛋白的热图。通过蛋白质-蛋白质相互作用（PPI）网络可以识别与NTDs相关的主要通路，如刺猬、Wnt、p53和河马信号通路以及叶酸介导的一碳代谢池。还发现NTDs中上调的核糖体蛋白如RPL13和RPL14的调控对神经管发育有一定影响。我们的结果揭示了低叶酸诱导的NTD小鼠组织中的蛋白质组变化。验证表明核糖体蛋白在NTDs发育过程中起调节作用，并为NTDs的发病机制和预防措施提供了新思路。
