PM2.5致先心病中DNA甲基化调控及其作用机制

Congenital heart disease (CHD) is the leading cause of birth defects in China. Increasing evidence links the atmospheric fine particulate matter (PM2.5) exposure during pregnancy to congenital heart disease, but the mechanisms remain to be elucidated. We previously reported that PM2.5 activated AhR and induced DNA methylation alterations, leading to heart defects in zebrafish embryos. Further studies in human embryonic stem cells showed that Serca2A was downregulated companying with DNA hypermethylation in promoter region, while overexpression of Serca2A alleviated the inhibitory effects of PM2.5 on cardiac differentiation. Thus, DNA methylation plays an essential role in PM2.5-induced CHD. We hypothesize that PM2.5 modulates DNA methylation via AhR, and DNA methylation changes further inhibit the expression of Serca2A, leading to abnormal heart development. In this project, human embryonic stem cells and transgenic zebrafish embryos will be used as models to establish a cardiac-specific analysis platform. Techniques such as gene silencing, gene overexpression and immunoprecipitation will be applied to identify the molecular pathways. Live cell imaging system and electrophysiology methods will be used to examine the physiological functions. The aim of this project is to explore the effects and regulation mechanisms of DNA methylation in the cardiac developmental toxicity of PM2.5, which will provide scientific basis and molecular targets for the prevention and treatment of air pollution-induced CHD.

我国新生儿出生缺陷中先心病居首位，有证据表明PM2.5孕期暴露与先心病相关，但机制不明。我们前期报道了PM2.5激活多环芳烃受体(AhR)，影响DNA甲基化水平，导致斑马鱼胚胎心脏畸形。而后又发现PM2.5引起人胚胎干细胞Serca2A启动子区DNA高甲基化及表达降低，过表达Serca2A能缓解PM2.5对心肌分化的抑制。因而，DNA甲基化在PM2.5致先心病中发挥重要作用。我们提出假设：PM2.5通过AhR引起DNA甲基化异常，而DNA甲基化变化进一步影响Serca2A表达，导致心脏发育障碍。本研究将结合人胚胎干细胞和转基因斑马鱼模型建立心肌特异性分析平台。采用基因沉默、过表达、免疫共沉淀等方法明确分子途径，以活细胞工作站成像和电生理等方法检验生理学功能。本课题旨在探索PM2.5心脏发育毒性中DNA甲基化调控及其作用机制，为预防和治疗大气污染引起的先心病提供科学依据和分子靶标。

先天性心脏病（先心病）的发病率在我国新生儿出生缺陷中位居首位，越来越多的证据表明包括大气污染在内的环境因素是引起先心病的重要原因。国内外多项流行病学研究显示PM2.5与胎儿心脏发育异常具有相关性。大气PM2.5暴露可增加自发流产率，考虑到心脏发育异常是引起流产的重要因素，所以PM2.5对心脏发育的实际危害可能远比流行病报道更为严重。本课题旨在评估PM2.5的心脏发育毒性，研究其毒性机制，探索相应的干预手段。该研究利用模式生物斑马鱼胚胎发育模型，结合人胚胎干细胞心肌定向分化模型，围绕DNA甲基化及其下游效应，针对PM2.5致心脏发育缺陷开展研究。本课题从细胞增殖，分化，氧化应激，心功能等方面进行毒性评价；从基因表达调控，DNA甲基化修饰水平等层次探讨PM2.5对心脏发育毒性的作用机制。该研究发现PM2.5提取物：①导致斑马鱼心脏发育畸形率升高，心功能降低；影响人胚胎干细胞来源的心肌细胞体外正常搏动；②显著激活多环芳烃受体信号通路；③引发全基因组范围内DNA甲基化水平异常变化；④引发甲基化变化的敏感基因主要募集在胚胎发育，细胞分化，DNA复制及修复等方面；⑤显著降低甲基化供体与受体比值SAM/SAH；⑥影响Serca2A，P21等基因启动子区域及基因本体区域甲基化调控序列修饰变化；⑦引发内质网应激及线粒体生成和产能障碍，介导心脏发育毒性。进一步，该课题从干预方面证实：①叶酸可有效缓解PM2.5导致的心脏组织DNA甲基化异常波动；②叶酸和白藜芦醇联合使用可有效逆转PM2.5心脏毒性作用。该项目从表观遗传层面阐述孕期PM2.5致先心病的分子机制，为保护干预提供分子靶标。