左归丸孕期给药预防子代大鼠IGT的表观遗传学机制

The theory of Developmental Origins of Health and Disease (DOHaD) holds that if life experienced adverse factors (dysfunction of uterus and placenta, malnutrition, etc.) in the early stages of development, including fetal period, the risk of getting developing chronic diseases such as diabetes, obesity, cardiovascular and cerebrovascular diseases will increase in adulthood. Impaired Glucose Tolerance (IGT) is also related to this theory, and is a common pre-pathological stage of above diseases. We compared the consistency and similarity between the theories of "kidney governs reproduction and development", "congenital essence and acquired essence nourish each other" in traditional Chinese medicine at molecular level and the mechanisms of life development, heredity and epigenetic modification. Based on this scientific hypothesis, we found that the Bushen Formula, Zuogui Pill, fed during pregnancy can effectively reduce the risk of IGT in offspring, and this effect is achieved by modifying and regulating the expression network or transcriptome of IGT-related genetic susceptibility genes in early development. And this project has been accepted by the Ministry of Science and Technology. The purpose of this study is to compare the offspring of rats fed with high-sugar and high-fat diet whose mother are stimulated by adverse factors during pregnancy and those offsprings fed with Zuogui Pill at the same time during pregnancy, and to find the epigenetic mechanism of zuogui pill fed during pregnancy to prevent IGT of offspring rats, so as to provide a scientific basis for creating a new method of promoting health and preventing diseases based on the theory of DOHaD from the perspective of traditional Chinese medicine.

健康与疾病的发育起源(DOHaD)理论认为，生命在包括胎儿期在内的发育早期阶段经历不利因素（子宫胎盘功能不良，营养不良等），将会增加其成年后患糖尿病、肥胖、心脑血管疾病等慢性疾病的风险。糖耐量低减(IGT)也与这一理论相关，且是上述疾病共同的一个前期病理阶段。比较了中医学“肾主生殖发育”、“先后天精气相互滋养”等理论在分子水平与生命发育、遗传及表观遗传修饰机制的一致性和相似性，并基于这一科学假说，我们发现在动物孕期予补肾方左归丸干预能有效降低其子代发生IGT的风险，并且这一作用是通过在发育早期修饰和调节相关遗传易感基因表达网络或转录组实现的。且这一项目已经国家科技部验收。本研究拟将母代孕期受不良刺激，并高糖高脂饮食喂养的子鼠与孕期同时灌左归丸孕鼠的子代比较，从表观遗传学方面研究左归丸孕期给药预防子鼠IGT的机制，为从中医角度创造基于DOHaD理论促进健康和预防疾病的新方法提供科学依据。

本项目旨在以现代医学“健康与疾病的发育起源(DOHaD)”理论与中医学“健康与疾病的先天禀赋（NEHaD）”理论的交叉融合研究及前期研究为基础，以补肾方左归丸对糖耐量异常孕鼠进行灌胃，以观察其对母代及子代的影响，并对子代肝脏进行基因甲基化检测，以进一步阐明左归丸孕期给药预防糖耐量异常孕鼠子代大鼠糖耐量低减（IGT）可能的表观遗传学机制。.首先我们用左归丸高中低剂量（10g·kg-1、5g·kg-1、2.5g·kg-1），验证了左归丸对妊娠糖尿病（GDM）孕鼠有一定治疗作用，并且随剂量增加更明显。并证实其治疗作用可能与其改善孕鼠胰腺十二指肠同源盒因子-1（PDX-1）蛋白及mRNA的表达水平有关。.因研究团队前期研究已经证实左归丸孕期给药可预防子代成年期IGT的发生，为避免重复研究，将对子代的研究提前至胚胎期及刚出生阶段。由于发现左归丸对母代PDX-1有影响，故也对子代胎鼠的PDX-1进行了研究，发现左归丸亦可通过上调胎鼠胰腺发育调控因子HNF-6基因甲基化及其mＲNA表达，从而上调PDX-1蛋白表达，调节胎鼠胰腺的发育，且低剂量即可对HNF-6基因甲基化产生影响。故以左归丸低剂量孕期给药，进一步对出生7日龄子代肝脏进行全基因组甲基化检测，发现左归丸的保护作用可延续至子代出生后，可调节子代肝脏与胰岛素抵抗相关的PI3K/AKT通路的甲基化水平。这些可能是左归丸于糖代谢异常大鼠孕期给药预防子代成年期IGT的表观遗传学机制之一。这就从中医补肾角度为IGT的预防研究提出了新的研究思路与方法，并提供了进一步的实验依据。从而也为从胚胎期进行干预以预防子代糖代谢异常等慢性病药物的开发应用奠定了一定的理论基础。