六味地黄丸对2型糖尿病代谢网络调节及作用机制研究

Liu-Wei-Di-Huang Pill as a classical Traditional Chinese compound medicament, has been widely used in clinical practices to treat Diabetic Mellitus. Traditional Chinese Compound has lots of advantages based on comprehensive regulation actions in disease treatment. Meanwhile, the multi-components and multi-targets characters of Traditional Chinese medicine, make that it is hard to estimate their therapeutic effects and study their mechanisms. In this project, we proposed the hypothesis that using the metabolism network regulations to estimate Liu-Wei-Di-Huang Pill therapeutic effects and mechanism in Type 2 diabetic mellitus. This project plans to carry out the metabolomics research of Liu-Wei-Di-Huang Pill in Type 2 diabetic mellitus treatment. GC-MS and UPLC-Q-TOF were used to profile these serum samples which obtained from diabetic mellitus model and Liu-Wei-Di-Huang cure model. Systematic research the metabolites differences in diabetic mellitus model and control model, and the changes in metabolism network caused by Liu-Wei-Di-Huang in Type 2 diabetic mellitus treatment. In order to discover the biomarkers which can indicate the disease status and Ccompound therapeutic effects, the advanced pattern recognize method and canonical correlation analysis will be used to discover the biomarkers from these metabolism network. Therefore, we can use the endogenous metabolites in serum samples to estimate the therapeutic effects of Liu-Wei-Di-Huang pill. Furthermore, combining the clinical biological index and bio-information analysis to illuminate the mechanism of Liu-Wei-Di-Huang pill in type 2 diabetic mellitus treatment.

六味地黄丸作为经典中药方，被广泛用于临床糖尿病治疗。但中药多成分、多靶点的特点，传统方法对中药治疗作用评估和作用机制研究有一定难度。本项目提出利用六味地黄丸对2型糖尿病代谢网络调节来评估其作用效果和作用机制研究的假设。拟开展六味地黄丸对糖尿病治疗的代谢组学研究，采用GC-MS和UPLC-Q-TOF对建立的糖尿病模型和经六味地黄丸治疗后模型中各血清样本内源性代谢物进行检测；系统研究正常动物和糖尿病相关动物模型代谢产物差异，并对给药前后模型样品的代谢物差异进行系统分析，观察糖尿病发生、发展与经治疗后代谢网络变化；通过模式识别和相关性分析在代谢网络水平上筛选出能够监控疾病的代谢物和反映对糖尿病治疗效果的内源性生物标志物；基于显著性生物标志物进行代谢通路分析，并构建代谢网络；从生理生化指标的改变与代谢网络的生物分析，明确六味地黄丸在糖尿病治疗中作用机制，为指导临床用药、提高疗效提供客观依据。

六味地黄丸作为治疗糖尿病的经典中药方剂，已被证实具有调节糖代谢、调节免疫功能、减低肝葡萄糖-6-磷酸酶活性、降低糖尿病动物的血糖、血脂、游离脂肪酸并促进胰岛素的分泌等作用，但是其治疗糖尿病的具体作用机制依然不明确。因而，本项目通过代谢组学方法开展糖尿病发病机制和六味地黄丸治疗糖尿病的作用机制研究，主要开展的研究内容有：.（1）通过构建MKR转基因的糖尿病小鼠模型并给予不同剂量的六味地黄丸治疗，发现六味地黄丸能够降低糖尿病小鼠的血糖值和尿蛋白含量，证实六味地黄丸能够用于治疗糖尿病并改善糖尿病对肾脏等器官的损伤；.（2）通过GC-MS代谢组学研究发现，相比于正常组，糖尿病小鼠模型组中氨基酸、脂肪酸类代谢产物含量明显升高，在给予六味地黄丸治疗后，棕榈酸、硬脂酸等饱和脂肪酸类化合物含量得到有效回调；.（3）进一步通过网络药理学研究与分子对接技术，发现六味地黄丸中的槲皮素、山奈酚、薯蓣皂苷、Hancinone C、常春藤皂苷元、Garcinone B、Isofucosterol等成分可参与AGE-RAGE等信号通路的调节，进而激活PI3K/AKT1、TNF/NF-κB等通路发挥抗炎、抗氧化应激和减轻β细胞损伤的功效，在T2DM及其并发症（肾病、动脉粥样硬化）中发挥治疗作用；.（4）通过Goto-Kakizaki（GK）大鼠构建非肥胖型的糖尿病大鼠模型，并结合代谢组学和微生物组学开展糖尿病大鼠肠道菌群和粪便代谢产物分析，研究发现糖尿病大鼠菌群多样性、丰度等均存在显著改变；与糖脂代谢相关的菌群，如拟杆菌和变形菌门，也发生了显著变化；并且进一步在大鼠粪便中证实甘油磷脂等脂肪酸及其代谢产物含量要高于正常组。.通过系列研究，得到以下结论：六味地黄丸可通过调节糖尿病的糖脂代谢，从而达到治疗、改善糖尿病的效果。研究进一步发现在大鼠中与脂质代谢相关的菌群也发生显著变化，那么六味地黄丸是否能够通过干预肠道菌群而达到其治疗作用呢？这为本项目进一步研究提供依据。