宫内编程介导的微囊藻毒素产前暴露诱发的神经发育损伤研究

The occurence of toxic blooms of cyanobacteria has become a serious public enviromental and health problem throughout the world. Microcystins (MCs) are the most commonly documented cyanotoxins, with a range of molecular weights from 900 to 1100 Da. So far, more than 100 congeners of MCs have been indentified, among which microcystin-leucine-arginine (MC-LR) is the most abundant and toxic variant. Ecological and experimental studies have indicated that prenatal exposure to MCs delayed body development and induced congenital defects, but the exact mechanism is still little known. The aim of this study is to investigate the intrauterine programming alteration mechanism underlying behavioral impairment caused by prenatal MC-LR exposure. Prenatal rats are intraperitoneally administered with various doses of MC-LR from gestational day 10 (GD10) to GD19, maternal and fetal oxidative stress, placental damage, MC-LR accumulation in maternal and offspring, postnatal dysfunction in physical and mental development as well as intrauterine programming alteration of the HPT axis will be detected. We also measure the effects of hippocampal morphology of axon and myelin induced by prenatal MC-LR exposure. The expression of hippocampal axonal development and myelin associated protein are tested by Western blotting. Prenatal xenobiotic exposure alters the epigenetic modifications in the fetal developmental process in many ways including DNA methylation, thus affecting the normal development of fetus. Epigenetic phenomena are also involved in the intrauterine programming caused by MC-LR. Gene-specific methylation of DNA isolated from brains of rat offspring is assessed in our study. By which, the relationship between the differential methylation status of these genes and behavioral induced by prenatal MC-LR exposure will be discussed. Our results will elucidate the molecular mechanisms in developmental toxicity of MC-LR from a new perspective and provide defined targets for new strategies, preventing and ameliorating the harzards of MCs.

蓝藻水华的频繁发生已成为日益严重的环境和公共健康问题。微囊藻毒素（MCs)是危害最大的蓝藻毒素，其中以MC-LR的毒性最强。生态学调查及实验室研究证实母源性MC-LR暴露能够损伤子代发育并导致子代的神经行为异常，然而发生机制尚不明了。本项目采用孕鼠腹腔注射MC-LR模拟产前暴露，从母体、胎盘和胎儿三方面，系统检测MC-LR诱发的母体与胎儿的应激反应、胎盘损伤、母体与子代脑内的毒素蓄积及HPT轴的编程改变，观察MC-LR对仔鼠海马突触和髓鞘相关蛋白表达的影响及对其形态学的损伤，测定子代早期生理和神经行为发育的变化。环境毒素的孕期暴露会通过表观遗传修饰的改变引起胚胎发育的异常，表观遗传修饰成为宫内编程的重要机制，本研究重点关注DNA甲基化修饰在MC-LR产前暴露诱发的子代神经行为异常中的影响。通过本研究，我们将从新的视角阐述MC-LR神经发育毒性的分子机制，为防治MCs的毒性提供新的靶点。

蓝藻水华的频繁发生已成为日益严重的环境和公共健康问题。微囊藻毒素（MCs）是危害最大的蓝藻毒素，其中以MC-LR的毒性最强。生态学调查和实验室研究均证实MC-LR暴露能够损伤子代发育，然而发生机制尚不明确。在本项目中，通过对孕6-19天的小鼠皮下注射不同剂量的MC-LR探讨母源性藻毒素暴露对子代发育的影响及机制。实验结果显示，MC-LR能够在子代体内蓄积，损伤子代的生长发育和神经行为，同时诱导子代海马区神经元的内质网应激反应和细胞凋亡。MicroRNA表达谱芯片结果及后续的RT-PCR验证实验显示，miR-181a-5p的表达量在MC-LR产前暴露组子代脑内显著下调。生物信息学分析发现重要的内质网应激蛋白Grp78是miR-181a的调控位点。后续验证实验表明MC-LR可以通过抑制miR-181a的表达上调Grp78的表达水平介导MC-LR产前暴露诱发的子代脑内的内质网应激反应和认知损伤。表达谱芯片分析表明甲状腺激素信号通路下调，采用ELISA试剂盒检测子代甲状腺激素含量发现MC-LR的暴露可以干扰母体和子代的甲状腺激素信号。因此，我们的实验结果表明子代脑内的毒素蓄积、内质网应激反应、表观遗传修饰的变化和HPT轴的编程改变是MC-LR产前暴露诱发子代神经行为异常的重要介导机制。同时，针对评审人的建议，我们增加了MC-LR暴露对斑马鱼生长发育的影响。研究结果表明，MC-LR能够通过影响WNT信号通路抑制斑马鱼的生长。同时，MC-LR具有促肝脏肿瘤生成的效应，蛋白磷酸酶PP2A和PRMT5是重要的介导分子。我们的研究从新的视角阐述了MC-LR发育毒性的分子机制，为防治MCs的毒性提供了新的靶点。