海马GAD67宫内编程改变介导孕期尼古丁暴露致子代HPA轴高应激敏感性的发生机制

Chronic adult diseases (neuropsychiatry disorder and metabolic diseases) have a fetal origin, and high stress sensitivity of hypothalamic-pituitary-adrenal (HPA) axis is the major cause. However, the mechanism involved in the high sensitivity of HPA axis has been not reported yet. Previous studies in our lab demonstrated that prenatal nicotine exposure can cause a permanent change of set point and sensitivity in hypothalamic paraventricular nucleus (PVN) and cause high sensitivity of HPA axis in offspring. Recently, we found that nicotine can increase the expression hippocampal GAD67 in fetal rats and also the adult rats. Based on literatures, we hypothesized that nicotine can combine with hippocampus a4b2nAChR and induce the epigenetic alteration of GAD67 promoter and enhance the expression of GAD67. The enhanced expression of hippocampus GAD67 can decrease the Glu-GABA synaptic connections in the hypothalamus PVN-projecting region, and increase the sensitivity of PVN. Moreover, the epigenetic alteration and enhanced expression of hippocampus GAD67 can continue to after birth, and contribute to the high sensitivity of HPA axis in offspring. In the present study, we will use lab animals to confirm the important role of hippocampus GAD67 intrauterine programming alteration in the high sensitivity of HPA axis induced by prenatal nicotine exposure. Meanwhile, we will clarify the epigenetic mechanism for the enhanced expression of fetal hippocampus GAD67 in cellular and molecular levels, to clarify the introuterine mechanism of high sensitivity of HPA axis induced by prenatal nicotine?exposure.

成年慢性疾病（如神经精神性疾病和代谢性疾病）具有宫内起源，HPA轴高应激敏感性是其主要诱因。然而，HPA轴高敏感性的发生机制至今尚未明了。我们近期发现，孕期尼古丁暴露可升高子代大鼠出生前、后下丘脑PVN兴奋性，引起成年HPA轴高敏感性变化；进一步发现，子代大鼠出生前、后海马GABA合成限速酶GAD67表达也增加。综合文献，我们推测：尼古丁与其受体a4b2nAChR结合，引起胎海马GAD67表观遗传修饰及表达改变，继而减弱海马对胎下丘脑PVN-projecting区Glu-GABA的突触联系，使PVN兴奋性增加。这些改变可延续至出生后，是引起成年HPA轴高敏感性的重要原因。本项目拟在整体水平证实海马GAD67宫内编程改变在孕期尼古丁暴露致子代HPA轴高敏感性中的核心作用，进一步从细胞水平研究尼古丁上调胎海马GAD67表达的表观遗传机制，以阐明孕期尼古丁暴露所致HPA轴高敏感性的宫内发生机制。

成年慢性疾病（如神经精神性疾病和代谢性疾病）具有宫内起源，下丘脑-垂体-肾上腺（HPA）轴高应激敏感性是其主要诱因。我们前期发现，孕期尼古丁暴露可引起成年子代大鼠HPA轴高应激敏感性变化，但其宫内编程机制不清。通过本项目研究，我们发现孕期尼古丁暴露可引起成年子代大鼠下丘脑局部谷氨酸能/伽马氨基丁酸（Glu/GABA）能神经输入的失衡和潜在兴奋性增强的编程性变化。此外，孕期尼古丁暴露可致成年子代海马谷氨酸脱羧酶67（GAD67）表达上调，伴随GAD67启动子−1019至−689区域的DNA甲基化水平降低、DNA甲基转移酶1（Dnmt1）表达抑制、海马局部GABA含量增加和GABA能神经元数目增加。进一步发现，孕期尼古丁暴露胎鼠亦存在与成年子代的一致性改变。体外实验发现，不同浓度尼古丁可直接引起大鼠胎海马神经元H19-7细胞系GAD67表达上调、GAD67启动子DNA甲基化水平降低和Dnmt1表达抑制，以及GABA能神经元数目增加，且这些改变可被特异性胆碱受体a4b2nAChR的抑制剂—DHbE所逆转。我们认为，孕期尼古丁暴露会增加子代大鼠下丘脑的潜在兴奋性，这可能会永久地改变下丘脑的调定点并进一步诱导成年后HPA轴高敏感性发生。其机制可能与海马GAD67表达上调性编程改变促使海马中Glu向GABA转化，进一步减弱对HPA轴的负反馈调节功能有关；尼古丁诱导的海马GAD67表观遗传修饰异常可能是HPA轴高应激敏感性重要分子机制。同时基于本项目的拓展研究，我们证实孕期外源物（尼古丁、乙醇、咖啡因）暴露所致子代HPA轴高应激敏感性存在共性现象，孕期外源物暴露可能通过直接效应介导子代大鼠海马神经元兴奋性损伤和GAD67代偿性上调，GAD67及其表观遗传标记可考虑作为HPA轴相关神经精神性疾病易感的早期标志物。这对阐明HPA轴高应激敏感性的宫内编程机制，为胎源性神经精神性疾病的早期预警技术提供了研究依据，同时也为早期有效防治胎源性神经精神性疾病提供了可能。本项目在药理与毒理学权威杂志Arch Toxicol、Toxicology、Toxicol Lett等发表第一/通讯作者SCI论文7篇，中文核心3篇，参加国内外学术会议11次，申请专利1项。