基于NMDA受体基因表观遗传修饰研究母源双酚A暴露致雄性仔鼠学习记忆降低的分子机制

Bisphenol A (BPA), one of the most common endocrine-disrupting chemicals, has been reported to adversely impact the learning and memory. Our previous study found that maternal BPA exposure impaired learning-memory by concomitant down-regulation of N-methyl-D-aspartate receptor subunit 1 of hippocampus in male offspring rats, but the molecular mechanisms underlying BPA interacts within the NMDA receptors is unclear. Studies have shown that DNA methylation and histone acetylation can influence each other, they may jointly regulate the expression of genes related to cognition and affect cognitive processes. Accordingly, we propose the hypothesis that epigenetic modifications of NMDA receptors gene expression may be involved in the molecular mechanism of maternal BPA exposure on impairment of learning and memory in male offspring rats. In order to confirm the hypothesis, in the present study, we examine the effects of developmental exposure through maternal diet to 50mg BPA/kg at postnatal day 21. Using the open field, object recognition test and Morris water maze, we examine the effects of maternal exposure to BPA on learning and memory in male offspring; applied diverse epigenetic experimental methods, this study observe the changes of the male offspring hippocampus NMDA receptors (NMDAR1, NMDAR2A, NMDAR2B) gene alterations in promoter DNA methylation and histone deacetylation, in order to illustrate the toxicity mechanism of epigenetic modifications of the NMDA receptors to explore maternal BPA exposure on impairment of learning and memory in male offspring rats, to provide the theory basis for research of BPA neurotoxicity.

双酚A（Bisphenol A，BPA）是一种广泛存在的环境内分泌干扰物，可以损害学习记忆功能。本课题组前期研究发现母源BPA暴露可通过下调NMDA受体的表达而降低雄性仔鼠学习记忆能力，但BPA下调NMDA受体的具体分子机制尚不清楚。研究表明DNA甲基化和组蛋白乙酰化相互影响，可共同调控认知相关基因的表达而影响认知过程。据此，本课题提出“NMDA受体基因的表观遗传修饰可能参与母源BPA暴露致雄性仔鼠学习记忆降低的分子机制”这一假说。为证实该假说，本课题构建母源BPA暴露模型，运用学习记忆行为学和表观遗传学等实验技术，着重研究海马内微量注射DNA甲基转移酶抑制剂和组蛋白去乙酰化酶抑制剂前后，BPA暴露对雄性仔鼠学习记忆行为和海马NMDA受体基因启动子区DNA甲基化和组蛋白去乙酰化的影响，旨在阐明NMDA受体基因表观遗传改变致BPA损害学习记忆的毒性机理，丰富BPA的神经毒性研究理论。

双酚A（Bisphenol A，BPA）是一种广泛存在的环境内分泌干扰物，可以损害学习记忆功能。本课题组前期研究发现母源BPA暴露可通过下调NMDA受体的表达而降低雄性仔鼠学习记忆能力，但BPA下调NMDA受体的具体分子机制尚不清楚。研究表明DNA甲基化可调控认知相关基因的表达而影响认知过程。据此，本课题提出“NMDA受体基因的表观遗传修饰可能参与母源BPA暴露致雄性仔鼠学习记忆降低的分子机制”这一假说。为证实该假说，本课题构建母源BPA暴露模型，运用水迷宫行为学和分子生物学实验技术，着重研究BPA暴露对雄性仔鼠学习记忆行为和海马NMDA受体基因启动子区DNA甲基化的影响，结果显示：（1）与对照组相比，母源BPA暴露的雄性仔鼠平台逃避潜伏期明显延长，水迷宫测试阶段在目标象限的探索时间和穿越平台次数显著减少，即母源BPA暴露损害了子代雄性大鼠的空间学习记忆能力；（2）BPA暴露显著下调了雄性仔鼠海马NMDARs的基因和蛋白表达水平；（3）亚硫酸氢盐测序分析显示，NMDAR1启动子区甲基化水平增加，这与母源BPA暴露后雄性后代海马中NMDAR1mRNA表达降低一致；（4）BPA暴露的子代雄性大鼠海马DNA甲基化酶DNMT3a和DNMT3b基因和蛋白表达均上调，BPA中剂量暴露组即BPA-40增加了海马DNA去甲基化相关酶TET1、Gadd45a和Gadd45b的基因表达水平；（5）转录组测序结果显示母源BPA暴露影响了子代雄性大鼠海马转录组mRNA的表达。这些研究数据表明BPA引起的空间学习记忆能力降低可能与海马区相关学习记忆重要基因的表观遗传修饰变化有关。本研究将为BPA安全剂量标准的制定提供参考依据，并丰富BPA的神经毒性研究理论。