Effects And Mechanisms Of Ampelopsis Grossedentata And Dihydromyricetin On Preventing Obesity In Mice

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 downregulated 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.