BDNF-TrkB-ERK通路在妊娠期铅和精神压力复合暴露影响子代认知发育中的作用机制研究

Recently, increasing attention has been paid to the effects of the combined prenatal lead and stress exposure on fetal health. In addition to exogenous lead exposure, pregnant women are susceptible to endogenous lead exposure (due to the mobilization of lead from maternal bone stores into circulation during pregnancy) and special stress during pregnancy. Our preliminary human study showed that, compared with toddlers with low maternal stress during pregnancy, the adverse association between prenatal lead exposure and toddlers’ cognition was stronger among toddlers with high maternal stress during pregnancy. Based on our animal studies and literature review, a single prenatal exposure to lead or stress would impair learning and memory, the hippocampal microstructure and expression of brain-derived neurotrophic factor (BDNF) in young rats, and a synergistic impairment in pups’ neurodevelopment caused by such combined prenatal exposure had been observed. However, until now, there are no other epidemiological studies focusing on neurotoxic effects of the combined exposure, and no studies are available to explore the role of BDNF-TrkB-ERK pathway in the impacts of such combined prenatal exposure on the cognitive development in the offspring. .Therefore, we propose to use both epidemiological and basic studies to address the effects of the combined prenatal lead and stress exposure on the cognitive development: (1) We plan to follow our Shanghai Birth Cohort (SBC) study which has recruited 5000 pregnant women, to assess maternal stress levels during pregnancy based on related stress scales and serum cortisorone levels, and prenatal lead exposure levels based on maternal and cord blood lead levels. After the information of prenatal lead and stress exposure is collected, a nested matched subcohort will be established to focus on the mother-child pairs with the combined prenatal lead and stress exposure, and their counterparts (the prenatal lead exposure group, the prenatal stress group and the control group, each group will contain 360 mother-child pairs). The cognitive development in the offspring will be followed up using Ages &Stages Questionnaires at 6 and 12 months old, using Bayley scales (the 3rd edition) at 24 months old. In addition, the protein, phosphorylated protein, and mRNA levels of BDNF, TrkB and ERK, and the general DNA methyltransferase level will be examined in cord blood. (2) The animal model with the combined prenatal lead and stress exposure will be established. Hippocampal injection of human-recombinant BDNF (hrBDNF) or hrBDNF plus U0126 (antagonist of ERK1/2) will be applied before and after the contextual fear conditioning test, and the short- and long-term memory will then be assessed. Novel object recognition test will then be performed to test the learning and memory of pups. Hippocampal microstructure will be examined using electron microscopy. The mRNA and protein expression levels of the key synaptic molecules, including synaptophysin, postsynaptic density protein 95 (PSD-95), and glial fibrillary acidic protein (GFAP) will be tested. Molecular biological analyses including real-time PCR, western blot, Elisa and bisulfite pyrosequencing (bisulfite pyrosequencing will be used to test DNA methylation levels at the promoter region of BDNF exon IV) will be applied to assess the roles of gene and protein expression and the phosphorylation and methylation regulation of BDNF-TrkB-ERK pathway in the developmental neurotoxicity due to the combined prenatal exposure..Our desire is, by this study, to explore the effects of the combined prenatal lead and stress exposure on the cognitive development, and to understand its underlying mechanism that mediates the effects of the combined prenatal lead and stress exposure on offspring’s cognitive development.

孕妇会遭受骨铅释放所致的内源性铅暴露和阶段性精神压力。胎儿未成熟脑对铅和压力的不良影响均敏感。我们前期研究发现，孕期铅和压力复合暴露诱发子代认知损伤可能有协同效应。但相关研究较少，且机制不明。根据前期结果和文献，我们提出脑源性神经营养因子(BDNF)-TrkB-ERK通路及该通路的磷酸化甲基化修饰可能参与该复合暴露的神经发育毒性。我们将①依托上海优生儿童队列，评估5000名孕妇的铅和压力复合暴露水平，选择复合暴露孕妇建立巢氏配对队列（复合、单独暴露及对照组孕妇各360例），测脐血BDNF、TrkB、ERK的基因蛋白、磷酸化和甲基化水平。随访婴儿6、12、24个月时的认知水平。②构建孕期复合暴露大鼠模型，恐惧实验前后海马注射hrBDNF或hrBDNF＋ERK阻滞剂，评估仔鼠学习记忆、海马超微结构、突触关键蛋白、及该通路的关键基因蛋白表达、磷酸化及甲基化水平。为探讨该复合暴露的防治提供新依据。

妊娠期铅和精神压力复合暴露是妊娠期非常常见的复合暴露，发育中的胎儿大脑容易受到出生前铅和过度精神压力复合暴露的影响。但针对该复合暴露的神经发育毒性作用的研究较少。本项目进行（1）人群研究：依托“上海优生队列”，从孕早或孕前招募孕妇，在孕早、中、晚、婴儿出生时、42天、6个月、12个月、24个月多次随访，评估孕早期和脐带静脉血血铅、妊娠期压力水平，及婴幼儿认知发育水平（6、12月用ASQ，24个月用贝利3），分析生命早期铅和压力单独及复合暴露对子代认知发育的影响。结果发现，孕早期（胚胎早期）和脐带血（出生前）血铅几何均数分别为14.5 ug/L和15.3 ug/L，妊娠期知觉压力、焦虑和抑郁阳性率总体较低。妊娠期压力和铅单独暴露与婴幼儿神经发育呈负相关。出生前铅压力复合暴露与婴幼儿神经发育多能区负相关但均未达统计学意义（回归系数大但可信区间也较大）。胚胎早期铅压力复合暴露与婴幼儿沟通、社交情绪能力负相关，尤其对24月龄幼儿社交情绪能力有明显负面影响 (P=0.016)。出生前铅、压力和复合暴露后脐带血清BDNF水平没有显著改变。（2）动物实验：将孕鼠分为孕期压力、铅、铅压力复合暴露组和对照组，运用 Morris 水迷宫检测子代幼鼠空间学习记忆水平，在条件恐惧实验前向幼鼠海马注射hr-BDNF、hr-BDNF+U0126等评估药物干预对幼鼠恐惧性学习记忆和消除的影响。透射电镜和 HE染色观察仔鼠海马形态学变化。运用 16s rRNA 测序比较幼鼠肠道菌群表达；检测子代大鼠海马BDNF-TrkB-ERK 通路mRNA蛋白表达水平和 p-ERK 水平。结果发现出生前铅暴露引起幼鼠血/骨铅升高，出生前压力会促进该效应；出生前铅-压力复合暴露损害学习记忆能力，破坏海马细胞形态结构，降低肠道菌群丰富度及均匀度，恐惧实验后BDNF-TrkB-ERK通路蛋白的表达水平上调，抑制对恐惧性记忆的消退能力，外源性补充hr-BDNF可能缓解恐惧反应。本项目提示妊娠期铅压力复合暴露对子代神经发育的不良影响高于单独暴露，胚胎早期复合暴露的影响可能更甚于出生前复合暴露。海马BDNF-TrkB-ERK 通路蛋白的表达水平变化和子代大鼠肠道菌群改变可能参与了影响机制。