肥胖，是由于食物摄入过多或机体代谢状态的改变而导致体内脂肪积聚过多造成体重过度增长并引起人体病理、生理改变的慢性代谢性疾病，是2型糖尿病、非酒精性脂肪性肝病、高血压、冠心病等疾病的高风险因素。目前全球约有22亿人超重，其中约7.12亿人（占到全球人口的10%）属于肥胖症。随着市面上减肥药物安全性事件的发生以及人们健康理念的变化，改善生活方式正逐渐成为对抗肥胖的关键手段，其中功能性食品，以茶饮为代表凭借其服用方便、活性较强且毒副作用较小的特点更易于被大众接受，主要发挥预防肥胖的作用。

藤茶（Ampelopsis grossedentata）是我国南方的民间传统用茶，含量最高的成分为双氢杨梅素，基于课题组前期工作基础及文献报道，藤茶及双氢杨梅素在预防糖脂代谢类疾病方面具有很大潜力，而藤茶缺乏与肥胖症相关的系统体内药效学研究，作用机制未有深入研究的报道。本研究旨在研究藤茶及主要成分双氢杨梅素预防肥胖的药效及作用机制，为后续相关的大健康产品研究开发提供科学数据支持。

本研究首先建立了以8周高脂饮食诱导的肥胖C57BL/6J小鼠模型，探究藤茶水提物及双氢杨梅素预防肥胖的药效，结果显示二者均降低肥胖小鼠的体重、体脂率，降低了血清胆固醇、甘油三酯水平，高胰岛素血症、高瘦素血症及白色脂肪组织的脂质堆积。为进一步研究其机制，我们采用转录组学的手段对小鼠皮下脂肪和棕色脂肪组织中显著变化的差异基因及富集通路进行分析，发现藤茶水提物可能主要通过抑制白色脂肪中的脂肪酸合成，调节棕色脂肪的产热过程发挥预防肥胖的作用。而双氢杨梅素影响了涉及脂代谢的多个通路，包括白色脂肪的细胞分化、脂肪酸的合成及摄取、甘油三酯的合成等及棕色脂肪的细胞分化及产热过程等。采用分子对接对双氢杨梅素可能的潜在靶点进行预测，结果显示PPARγ，C/EBPα，FABP4，UCP-1是与其相互作用较强的蛋白靶点，进一步分子动力学模拟结果表明，PPARγ、C/EBPα、FABP4、UCP-1与其在动态模拟中结合稳定。实时定量荧光PCR结果验证，双氢杨梅素可能通过PPARγ/C/EBPα/FABP4通路抑制白色脂肪的细胞分化、增加UCP-1的表达促进白色脂肪棕色化及棕色脂肪的产热过程发挥作用。这是首次从脂肪分化的角度阐述双氢杨梅素预防肥胖的作用。

基于双氢杨梅素预防肥胖C57BL/6J小鼠模型的研究，其发挥作用的主要靶器官为脂肪组织，然而该模型未形成高甘油三酯血症及肝脏的脂肪性病变，尚不能从整体反应机体的脂肪代谢情况。为进一步确认双氢杨梅素预防肥胖是通过脂肪代谢相关通路，继而采用瘦素基因缺陷的肥胖ob/ob模型小鼠，发现其干预12周后，ob/ob小鼠的体重、血脂水平、肝脏功能及肝脏、脂肪组织中的脂质蓄积情况均得到显著改善。因miRNA与白色脂肪的生成与分化密切相关，我们采用miRNA测序，首次发现了两个可能发挥作用的标志miRNA，miR-196a-2-3p、miR-15b-3p，其中miR-196a已有报道称能有效调节脂肪细胞分化，采用实时荧光定量PCR再次确认双氢杨梅素能通过PPARγ/CEBPα/FABP4信号通路有效抑制脂肪细胞分化，首次阐明双氢杨梅素发挥作用极可能与miR-196a-2-3p的差异化表达相关。此外，双氢杨梅素干预后ob/ob小鼠的高甘油三酯血症及脂肪肝病变得到显著改善，转录组学结果显示其可能通过调节肝脏的胆固醇代谢、胆汁分泌通路发挥作用，为寻找潜在的作用靶点，采用分子对接发现FXR，SREBP-1C，ACC，FAS可能是最有潜力，进一步分子动力学模拟验证FXR与其结合的稳定性，提示双氢杨梅素可能调节FXR介导的胆汁酸代谢、肝脏中脂肪的从头合成两条通路。对胆汁酸含量进行测定，发现肝脏中结合型胆汁酸含量升高，肝脏、粪便中石胆酸及其硫化物含量明显上升，结合实时荧光定量PCR、Western blot等验证手段发现，双氢杨梅素显著促进了肝脏中初级胆汁酸的结合和转运、抑制了回肠中胆汁酸的重吸收，同时促进了胆汁酸的排泄。这是首次发现双氢杨梅素通过FXR介导的胆汁酸代谢通路维持机体的胆固醇稳态。采用宏基因组的测序手段对双氢杨梅素干预后ob/ob小鼠的肠道微生物群进行种水平的测定，发现罗伊氏乳杆菌（Lactobacillus reuteri）的表达水平下调，参与到机体的代谢状态。此外，胆汁酸代谢核心因子FXR在肝脏中激活可以抑制下游的脂肪从头合成通路，关键蛋白SREBP-1C，ACC，FAS的表达量显著下降，从而改善高甘油三酯血症。

综上所述，本研究明确了藤茶及双氢杨梅素预防肥胖的巨大潜力，首次从以下几个方面阐释了双氢杨梅素的可能作用机制：1）通过PPARγ/C/EBPα/FABP4通路抑制白色脂肪的细胞分化，可能与miR-196a-2-3p的差异化表达相关；促进白色脂肪的棕色化、棕色脂肪的产热作用；2）调节FXR介导的胆汁酸代谢通路及肝脏中的脂肪从头合成通路，肠道菌群尤其是乳酸杆菌（Lactobacillus）发挥了重要作用。藤茶作为新资源食品及具有开发潜力的茶饮植物，我们对藤茶及主要成分双氢杨梅素预防肥胖、延缓代谢紊乱等相关进程的药效与机制研究，为其产品开发提供了数据支撑和科学依据，助力大健康产业。

Obesity is a chronic metabolic disease, caused by excessive food intake or changes of the metabolic state, resulting in excessive accumulation of fat, excessive weight gain and human pathological and physiological changes. Obesity is caused by genetic, environmental, psychological and social factors and it’s a high risk factor for type 2 diabetes, non-alcoholic fatty liver disease, hypertension, coronary heart disease and other diseases. Currently, there are about 2.2 billion overweight people in the world, of which about 712 million (10% of the global population) are obese. With the occurrence of safety incidents of weight-loss drugs on the market and changes in people's health concepts, improving lifestyles is gradually becoming a key means of combating obesity. Functional foods, represented by tea drinks, are easy to take and active and non-toxic, mainly presenting the activity of preventing obesity.

Vine tea (Ampelopsis grossedentata) is a traditional folk tea in southern China. Dihydromyricetin, has a content of up to 30% in vine tea. Based on researches and the foundation in our laboratory, vine tea has great potential in regulating glucose and lipid metabolic disorder. Howerer, in vivo pharmacodynamics studies of vine tea in preventing obesity were lacking and the mechanism has not been reported. The purpose of this study was to study the pharmacodynamics and main mechanism of vine tea and dihydromyricetin in preventing obesity, and to provide scientific data support for the subsequent research and development of related products.

In this study, an 8-week high-fat diet-induced C57BL/6J obese mouse model was established to explore the efficacy of water extract of vine tea and dihydromyricetin in preventing obesity. The results showed that both water extract of vine tea and dihydromyricetin decreased the body weight, body fat rat, and improved hyperlipidemia, hyperinsulinemia, hyperleptinemia and lipid accumulation in white adipose tissue. We used transcriptomics to analyze the significantly changed differential genes and enrichment pathways in mouse subcutaneous fat and brown fat, and found that the water extract of vine tea may mainly inhibit fatty acid synthesis in white fat, regulate the thermogenesis of brown fat. Dihydromyricetin affects multiple pathways involved in lipid metabolism, including cell differentiation of white fat, synthesis and uptake of fatty acids, synthesis of triglycerides, and cell differentiation and thermogenesis of brown fat. Then, molecular docking was used to predict the possible potential targets of dihydromyricetin. The results showed that PPARγ, C/EBPα, FABP4, and UCP-1 are protein targets that interact strongly with dihydromyricetin. Further molecular dynamics simulations showed that PPARγ and dihydromyricetin bind stably. Real-time quantitative PCR results confirmed that dihydromyricetin may inhibit the cell differentiation of white fat through PPARγ/C/EBPα/FABP4 pathway, and increase the expression of UCP-1 to promote the browning of white fat and the thermogenesis of brown fat. This is the first time to clarify the role of dihydromyricetin in preventing obesity from the perspective of white fat differentiation.

Based on the research on the prevention of C57BL/6J obesity mouse model by dihydromyricetin, the main target organ was adipose tissue. However, the model did not form hypertriglyceridemia and fatty lesions of the liver. In order to further confirm that dihydromyricetin prevents obesity through fat metabolism-related pathways, we then used leptin gene-deficient ob/ob mice, and found that after 12 weeks of intervention, the body weight, blood lipid levels, liver function, and lipid accumulation in liver and adipose tissue of ob/ob mice were significantly improved. Because miRNAs are closely related to the production and differentiation of white fat, we used miRNA sequencing to discover two marker miRNAs that may play a role, miR-196a-2-3p and miR-15b-3p for the first time. miR-196a has been reported that it can effectively regulate adipocyte differentiation. Therefore, real-time quantitative PCR was used to reconfirm that dihydromyricetin can effectively inhibit adipocyte differentiation may related with the differential expression of miR-196a-2-3p. In addition, after the intervention of dihydromyricetin, the hypertriglyceridemia and fatty liver lesions in ob/ob mice were significantly improved. Transcriptomic results showed that dihydromyricetin may play a role in regulating liver cholesterol metabolism and promoting bile secretion pathway. We used the molecular docking to looking for potential key targets and found that FXR, SREBP-1C, ACC, FAS may be the most potential targets, further molecular dynamics simulations verified the stability of FXR binding to dihydromyricetin, which suggested that it may interfere with two pathways of bile acid metabolism and de novo lipogenesis in the liver. The bile acid metabolomics results showed that the content of conjugated bile acids in the liver increased, and the content of lithocholic acid and its derivatives in the liver and feces increased significantly. Combined with real-time quantitative PCR and Western blot, it was found that dihydromyricetin significantly promotes the binding and transport of primary bile acids in the liver, inhibits the reabsorption of bile acids in the ileum, and promotes the excretion of bile acids. This is the first time to discover that dihydromyricetin maintains the body's cholesterol homeostasis through the FXR-mediated bile acid metabolism pathway. Metagenome sequencing was used to determine the species level of the gut microbiota of ob/ob and found that the expression level of Lactobacillus was down-regulated and involved in bile acid metabolism in the gut. In addition, activation of the core factor FXR in the bile acid metabolism can inhibit the downstream de novo lipogenesis pathway, and the expressions of key proteins, SREBP-1C, ACC, and FAS were significantly decreased, thereby improving hypertriglyceridemia.

In conclusion, this study clarified the great potential of vine tea and dihydromyricetin in preventing obesity, and explained the possible mechanism of dihydromyricetin for the first time from the following aspects: 1) Through the PPARγ/C/EBPα/FABP4 pathway inhibiting the cell differentiation of white fat which may be related to the differential expression of miR-196a-2-3p; promoting the browning of white fat, brown adipocyte differentiation and thermogenesis of brown fat; 2) Regulating the FXR-mediated bile acid metabolism pathway and de novo lipogenesis pathway in the liver. Gut microbiota, especially Lactobacillus, play an important role. Therefore, the study of efficacy and mechanism of vine tea and its main component dihydromyricetin in preventing obesity, delaying metabolic disorders and other related processes, provided data support for the product development and help the big health industry.