南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (6): 889-899.doi: 10.12122/j.issn.1673-4254.2023.06.03

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Notch信号通路抑制剂DAPT改善酒精诱导的斑马鱼神经元分化障碍

殷 果，李 荣，刘岳飞，王晓睛，吴炳义   

  1. 南方医科大学南方医院医学实验中心，广东 广州 510515；广州医科大学附属肿瘤医院放疗科，广东 广州 510095；聊城市人民医院神经内科，山东 聊城 252000
  • 出版日期:2023-06-20 发布日期:2023-07-07

Notch signaling pathway inhibitor DAPT improves alcohol-induced neuronal differentiation impairment in zebrafish

YIN Guo, LI Rong, LIU Yuefei, WANG Xiaojing, WU Bingyi   

  1. Medical Research Center of Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Department of Radiation Oncology, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou 510095, China; Department of Neurology, Liaocheng People's Hospital, Liaocheng 252000, China
  • Online:2023-06-20 Published:2023-07-07

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摘要： 目的 探讨Notch信号通路在胎儿酒精谱系障碍（FASD）斑马鱼模型的神经元分化及及感觉-运动能力中的作用。方法 首先，将斑马鱼胚胎分为二甲基亚砜（DMSO）组和50 μmol/L氮-[氮（-3,5-二氟苯乙酰基）-L-丙氨酰]-S-苯基甘氨酸丁酯（DAPT，Notch信号通路抑制剂）处理组，检测比较两组斑马鱼的死亡率、孵化率以及畸形率；比较两组斑马鱼的身体长度；利用原位杂交和qRT-PCR技术检测两组神经干/前体细胞标志物sox2、神经前体细胞分化因子neurogenin1、神经元标志物huc的表达；检测两组在无刺激、强光、震动刺激情况下的行为学表现。其次，将斑马鱼胚胎分为DMSO组、1.5%酒精处理组、DAPT处理组、酒精和DAPT联合处理组，利用原位杂交和qRT-PCR技术检测各组sox2、neurogenin1、huc的表达，检测Notch信号通路相关基因（Notch信号受体notch1a，促进神经发育的靶基因her8a及Notch胞内活性片段（NICD）的mRNA表达水平；检测各组在无刺激、强光、震动刺激情况下的行为学表现。结果 50 μmol/L DAPT处理斑马鱼胚胎后，受精后1 d（1 dpf）胚胎死亡率（P<0.01）显著增加，2 dpf孵化率显著降低（P<0.01），3 dpf畸形率（P<0.001）显著增加，15 dpf斑马鱼体长显著缩短（P<0.05）；斑马鱼胚胎sox2表达水平显著降低（P<0.01），neurogenin1（P<0.05）和huc mRNA（P<0.01）表达水平显著增加；在无刺激、强光、震动刺激情况下，DAPT处理组的斑马鱼运动距离均显著缩短（P<0.001），运动速度均显著降低（P<0.05）。单独使用1.5%酒精处理斑马鱼胚胎后，notch1a、her8a及NICD的mRNA表达水平显著升高（P<0.05）；1.5%酒精联合DAPT处理斑马鱼胚胎后，较酒精单独处理组，neurogenin1和huc mRNA表达水平显著增加，sox2 mRNA表达水平显著降低（P<0.01），在强光刺激下的运动距离显著变长，运动速度显著变快（P<0.05）。结论 酒精上调斑马鱼胚胎Notch信号、抑制神经元分化并降低感觉-运动能力，而抑制Notch信号通路可改善斑马鱼胚胎神经元分化及感觉-运动能力。

关键词: 酒精；胎儿酒精谱系障碍；神经元分化；Notch信号通路；DAPT

Abstract: Objective To explore the role of the Notch signaling pathway in regulating neuronal differentiation and sensorimotor ability in a zebrafish model of fetal alcohol spectrum disorder. Methods Zebrafish embryos treated with DMSO or 50 μmol/L DAPT (a Notch signaling pathway inhibitor) were examined for mortality rate, hatching rate, malformation rate, and body length at 15 days post fertilization (dpf). The mRNA expression levels of sox2, neurogenin1 and huc in the treated zebrafish embryos were detected using in situ hybridization and qRT-PCR, and their behavioral responses to strong light and vibration stimulation were observed. The zebrafish embryos were then exposed to DMSO, 1.5% ethanol, DAPT, or both ethanol and DAPT, and the changes in mRNA expression levels of sox2, neurogenin1, huc, and the Notch signaling pathway genes as well as behavioral responses were evaluated. Results Exposure to 50 μmol/L DAPT significantly increased the mortality rate of 1 dpf zebrafish embryos (P<0.01), decreased the hatching rate of 2 dpf embryos (P<0.01), increased the malformation rate of 3 dpf embryos (P<0.001), and reduced the body length of 15 dpf embryos (P<0.05). DAPT treatment significantly downregulated sox2 mRNA expression (P<0.01) and increased neurogenin1 (P<0.05) and huc (P<0.01) mRNA expressions in zebrafish embryos. The zebrafish with DAPT treatment exhibited significantly shortened movement distance (P<0.001) and lowered movement speed (P<0.05) in response to all the stimulation conditions. Compared with treatment with 1.5% ethanol alone, which obviously upregulated notch1a, her8a and NICD mRNA expressions in zebrafish embryos (P<0.05), the combined treatment with ethanol and DAPT significantly increased neurogenin1 and huc mRNA expression, decreased sox2 mRNA expression (P<0.01), and increased the moving distance and moving speed of zebrafish embryos in response to strong light stimulation (P<0.05). Conclusion Ethanol exposure causes upregulation of the Notch signaling pathway and impairs neuronal differentiation and sensorimotor ability of zebrafish embryos, and these detrimental effects can be lessened by inhibiting the Notch signaling pathway

Key words: ethanol; fetal alcohol spectrum disorder; neuronal differentiation; Notch signaling pathway; DAPT