孕期甜菜碱摄入通过调控PGC1α/PPARα信号通路基因甲基化缓解胎源性NAFLD的机制研究

Non-alcoholic fatty liver disease (NAFLD) of fetal origin is closely related to the gestational nutrition status, nutritional improvement in early life will alleviate the progression of NAFLD. Betaine is a kind of methyl donor naturally containing in the food, the lipotropic effect of betaine has been demonstrated in human and animal models with NAFLD, our previous studies have found that betaine could alleviate NAFLD by regulating DNA methylation of genes involved in PGC1 alpha/PPAR alpha signaling pathway. Based on our previous findings, we speculate: gestational betaine supplement could alleviate fetal original NAFLD, the underlying mechanism is associated with regulation on DNA methylation of genes in PGC1 alpha/PPAR alpha signaling pathway, however it has not been reported before. Firstly, we use gestational diabetes to induce fetal original NAFLD in the offspring, and explore whether gestational betaine supplement could relieve fetal original NAFLD; Secondly, based on vivo and vitro experiments, we confirm that the lipotropic effect of betaine is related to the regulation of DNA methylation and expression of genes in PGC1 alpha/PPAR alpha signaling pathway; Thirdly, we use human study to confirm the relationship between maternal betaine intake during pregnancy and the risk of fetal original NAFLD in the offspring. Our study may contribute to increase betaine intake for maternal population, and play an active role in preventing NAFLD since early life.

胎源性非酒精性脂肪肝病（NAFLD）与胚胎时期营养密切相关，生命早期营养改善将会减缓NAFLD的发生发展。甜菜碱是一种天然存在于食物中的甲基供体，其抗脂肪肝作用已在NAFLD人群和动物中得到验证，我们前期研究也发现甜菜碱可通过调节肝脏PGC1α/PPARα信号通路基因DNA甲基化缓解成年小鼠NAFLD。在此基础上我们推测：孕期甜菜碱摄入可缓解胎源性NAFLD，其机制与调控子代肝脏PGC1α/PPARα信号通路基因甲基化有关，但未见报道。因此，本研究首先采用妊娠糖尿病诱导胎源性NAFLD动物模型，观察孕期甜菜碱摄入缓解子代胎源性NAFLD的效果；并结合细胞实验确证甜菜碱是通过调节PGC1α/PPARα信号通路基因甲基化来发挥作用；最后利用人群研究验证孕期甜菜碱摄入与子代NAFLD发病风险的关系。本研究为孕产人群增加甜菜碱摄入提供实验依据，对从生命早期营养防治NAFLD具有积极作用。

子代非酒精性脂肪肝病（NAFLD）与母代肥胖和糖尿病密切相关。本课题组前期研究发现，甜菜碱可以通过调节肝脏脂代谢基因DNA甲基化来发挥抗脂肪肝作用，然而孕期甜菜碱摄入是否可以通过调控子代DNA甲基化来改善胎源性NAFLD，尚未见报道。本课题首先是收集了47例经肝脏病理诊断的NAFLD患者及18例对照人群，收集研究对象一般资料、血液和肝脏样本。结果显示，与对照组相比，NAFLD组肝脏全基因组DNA甲基化更低，血清同型半胱氨酸水平更高，甜菜碱/胆碱比值更低。肝脏全基因组DNA甲基化水平与肝脏炎症、纤维化和疾病进展呈现负相关。肝脏纤维化、疾病进展与血清同型半胱氨酸水平与呈现正相关，甜菜碱/胆碱比值呈负相关。人群实验结果预示甜菜碱及肝脏DNA甲基化参与了NAFLD的发生发展。另外，我们利用动物实验，对孕鼠进行高脂饮食+55mg/kg链脲霉素诱导孕期肥胖和高血糖模型，并用1%和2%甜菜碱进行干预，子代正常饮食喂养至8周后收集小鼠血液、肝脏样本。结果显示，在仔鼠中，孕期高脂饮食加链脲霉素可引起子代血清ALT、IL-6、TNFα和肝脏甘油三酯水平显著升高，肝脏病理显示脂质蓄积明显加重，而孕期甜菜碱补充可以降低其水平并缓解肝脏脂质蓄积。与对照组相比，模型组肝脏PGC1α和PPARαmRNA表达显著下降，2%甜菜碱补充则可上调其表达。模型组肝脏IL-18基因和ASC蛋白表达较对照组上升，孕期甜菜碱补充可以降低肝脏IL-18、ASC、CASP1、NLPR3和IL-1β基因和（或）蛋白表达。模型组肝脏PGC1α基因启动子区的-3685CpG位点的甲基化程度显著升高，PGC1α启动子甲基化水平与其mRNA表达呈负相关。甜菜碱干预后肝脏同型半胱氨酸和S腺苷同型半胱氨酸较模型组显著降低。相比于模型组，2%BET组DNMT1、DNMT3A与DNMT3B基因mRNA水平明显下降。动物实验表明孕期甜菜碱补充可缓解仔鼠胎源性NAFLD发生发展，可能与调节肝脏PGC1α/PPARα信号通路和NLRP3炎症小体相关基因表达和甲基化有关。本研究为孕期甜菜碱摄入预防子代NAFLD提供了相关的理论依据，也为指导孕期合理膳食来预防子代代谢性疾病提供一定参考。