基于AMPK信号通路的青钱柳多糖调控脂代谢相关基因DNA甲基化的研究

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is the key factor for the regulation of lipid metabolism in the liver, and is the centre for research of hyperlidemia and other metabolic syndromes. Epigenitics, regulated by nutritional and environmental factors, plays key role in the formation and development of the metabolic syndrome. Our previous studies showed that polysaccharide from Cyclocarya paliurus (Batal.) Iljinskaja had hypolipidemic effects, but the functional mechanism, especially the epigenitic mechanism is still unknown. Based on our previous researches, using hyperlipidemia rats as an animal model, the objective of the study will investigate the effect of Cyclocarya paliurus (Batal.) Iljinskaja polysaccharide on the gene expression and DNA methylation for those genes related to the lipid metabolism mediated by AMPK pathway at different growth stages and in different tissues with Bisulfite Sequencing PCR （BSP）and Quantitative real-time PCR (qRT-PCR) to analyze the relationships between gene expression and DNA methylation. Also, the immunohistochemical and computer image analysis techniques will be used to analyze the pathological features of the tissues after the polysaccharide intervention. Thus to reveal the relationship between hyperlipoidemia and the Cyclocarya paliurus (Batal.) Iljinskaja polysaccharide mediated by AMPK pathway at the level of molecule and epigenetics to elucidate the regulatory mechanism of the polysaccharide on the lipid metabolism and the targetted site of the co-regulatory function. The result of the study will establish a theoretical principle for studying and developing a new hypolipidemic functional factor and functional food, and provide a scientific basis for the further utilisation and development of the Cyclocarya paliurus (Batal.) Iljinskaja.

AMP激活蛋白激酶(AMPK)是肝脏脂质代谢调节的关键分子，是研究高脂血症及其他代谢综合征的核心。表观遗传修饰对代谢综合征的发生和发展产生着重要的作用，受营养和环境因素所调控。前期研究发现，青钱柳多糖具有降血脂作用，然而其作用的机制，特别是表观遗传学机制不清楚。本项目拟在前期工作基础上，以高脂血症大鼠为模型，采用BSP克隆测序法和qRT-PCR探讨青钱柳多糖通过AMPK信号通路调控脂代谢相关基因在不同阶段和组织中的DNA甲基化和表达水平，分析基因表达与DNA甲基化的关系；并采用免疫组化、计算机图像分析技术等检测技术分析多糖干预后组织的病理学特征，从而从分子水平和表观遗传学方面揭示青钱柳多糖通过AMPK及其信号通路与高脂血症的关系，进而阐明多糖调控脂代谢的机理，揭示共调控作用的关键作用靶点。本项目将为研究和开发新降脂功能因子及功能食品奠定理论基础，为青钱柳资源的深度开发和利用提供科学依据。

表观遗传修饰对代谢综合征的发生和发展产生着重要的作用，受营养和环境因素所调控。本项目拟在前期工作基础上，以高脂血症大鼠为模型，采用BSP克隆测序法和qRT-PCR探讨青钱柳多糖通过AMPK信号通路调控脂代谢相关基因在不同阶段和组织中的DNA甲基化和表达水平，分析基因表达与DNA甲基化的关系。结果表明，多糖处理可显著改善高脂血症小鼠血液和肝脏血脂水平、提高肝脏抗氧化状态。病理学研究表明，多糖可以改善肝脏和脂肪组织的病理学状态从而缓解肝脏和脂肪组织病变。多糖可提升高脂血症小鼠血清和肝脏中谷氨酸丙酮酸转氨酶（GPT）、SOD、GSH-Px等酶的活性，并降低MDA和脂质过氧化物含量，增强总抗氧化能力水平。同时能降低高脂血症小鼠肝脏中全基因组DNA 甲基化的水平，并且通过调节腺苷酸活化蛋白激酶（AMPK）信号通路、脂肪酸代谢通路、脂肪酸生物合成通路和脂肪细胞因子信号等通路，从而起到降脂作用。多糖可以显著降低高脂血症大鼠肝脏组织中LEPTIN、MC4R、MTTP、POMC、PPARγ五个基因启动子区DNA甲基化水平、显著降低脂肪组织中LEPTIN基因DNA甲基化水平，显著提高POMC基因DNA甲基化水平。Q-PCR结果表明，青钱柳多糖(CPP)能够显著下调高脂血症大鼠肝脏和脂肪组织中LEPTIN、MC4R、MTTP、PPARγ等基因的mRNA表达量，但对高脂血症大鼠肝脏和脂肪中POMC基因mRNA的表达量无显著的影响。多糖可显著抑制高脂血症大鼠脂肪中甲基转移酶（DNMTs）活性，相关性分析结果显示，LEPTIN、MC4R、MTTP、POMC的DNA甲基化水平与mRNA表达量无显著相关性，PPARγ基因呈现极显著相关，在肝脏和脂肪组织中LEPTIN、MC4R、PPARγ三个基因DNA甲基化水平与甲基转移酶活性呈显著正相关。结果可为青钱柳资源的深度开发和利用提供一定的科学依据。