Dihydromyricetin Improves Obesity And Insulin Resistance By Modulating The Composition Of Gut Microbiota

Obesity is a systemic chronic inflammatory disease,which is known as the main risk factor of metabolic diseases such as insulin resistance,type 2 diabetes and non-alcoholic fatty liver disease.Nowadays,unhealthy living and dietary habits including high-fat diet and chronic exercise lacking have become the primary causes of obesity and insulin resistance,where gut microbiota plays an important role during the process.The efficacy of traditional Chinese medicine on the prevention and treatment of obesity and insulin resistance associated with modulating of the gut microbiota composition is continually being confirmed,which has already attracted global attentions from different research areas.Ampelopsis grossedentata,also known as Vine tea,is a traditional Chinese medicinal and edible herb that possesses various efficacy including clearing heat and removing toxicity,inducing diuresis and reducing edema,calming the liver and lowering blood pressure,and promoting blood circulation and removing meridian obstruction.Dihydromyricetin（DMY）has been demonstrated as the primary active ingredient in vine tea with clinical application potential in prevention and treatment of obesity and insulin resistance,but the pharmacological effects and mechanisms still need to be further systematically investigated.Thus,for the first time,the present study aimed to demonstrate that DMY improves obesity and insulin resistance by modulating the composition of gut microbiota.The overall research content is divided into the following five chapters.Chapter 1:The pharmacokinetic study of DMY in ratsObjectives:Investigate the basic characteristics of pharmacokinetics of DMY after oral administration in rats.Methods:An LC-MS/MS method for the determination of DMY in rat plasma was developed and validated.The plasma concentrations of dextroisomer and racemate DMY over time after oral administration were determined using the developed LC-MS/MS method,and the pharmacokinetic parameters were analyzed.Results:The results of method validation study indicated that the developed LC-MS/MS method for the determination of DMY in rat plasma met the requirements of biological sample analysis.There were significant differences in the values of t_max（160±90.3 and36.7±41.7 min）,AUC_0-∞（42.8±5.92 and 32.0±9.03 mg×min/L）and Vd（1001±349.5and 1721±660.9 L/kg）between dextroisomer and racemate DMY,while no significant differences were found in the values of C_max,t_1/2,AUC_0-t,CL,MRT_0-t and MRT_0-∞.Conclusions:According to the overall results,both dextroisomer and racemate DMY were poorly absorbed into bloodstream and underwent rapid elimination processes after oral administration.Moreover,both dextroisomer and racemate DMY might highly distributed in tissues,which needed to be clarified in further tissue distribution and excretion study.Chapter 2:The tissue distribution and excretion study of DMY in rats.Objectives:Investigate the tissue distribution and excretion of DMY after oral administration in rats,which will provide theoretical basis for further pharmacological effects and mechanisms study.Methods:An LC-MS/MS method for the determination of DMY in rat tissues,urine and feces was developed and validated,and the concentrations of DMY in rat tissues,urine and feces over time after oral administration were determined using the developed LC-MS/MS method.Results:The results of method validation study indicated that the developed LC-MS/MS method for the determination of DMY in rat tissues,urine and feces met the requirements of biological sample analysis.After oral administration,the peak concentrations of DMY in different tissues were observed at 15 or 45 min,and the peak concentrations in stomach and small intestine were much higher than those in other tissues.After 45 min,the concentrations of DMY in tissues underwent a downward trend over time,which were reduced by approximately 90%of peak levels at 12 h.The urinary and fecal cumulative excretion ratios of DMY reached a plateau after 12 h.The final urinary cumulative excretion ratio was only 0.0145±0.0069%,while the final fecal cumulative excretion ratio could reach 18.95±6.87%.Conclusions:DMY could be distributed and eliminated rapidly in various tissues,and the most unconverted forms of DMY could be highly distributed in the gastrointestinal tract.The most unconverted forms of DMY were excreted in feces,while DMY was mainly excreted as metabolites.According to the present study and in vivo metabolic profile study done by our research group at the same period,the overall results demonstrated that the unconverted forms and metabolites of DMY were poorly absorbed into bloodstream.And both unconverted forms and metabolites of DMY were mainly excreted in feces through the intestinal tract.Chapter 3:The systematic evaluation of pharmacological effects and preliminary mechanism study of DMY on prevention and treatment of obesity and insulin resistance.Objectives:Systematically evaluate the preventive and therapeutic effects of DMY on high-fat diet-induced obesity and insulin resistance,and investigate the mechanism from the point of metabolic endotoxemia and systemic chronic low-grade inflammation resulted from the gut microbiota dysbiosis induced by high-fat diet.Methods:Obesity and insulin resistance mice induced by high-fat diet was treated with DMY at low,medium and high dose by intragastric gavage at the beginning or the end of animal model establishment,for the purpose of systematical evaluation of preventive and therapeutic effects of DMY on high-fat diet induced obesity and insulin resistance.On this basis,the levels of serum LPS,proinflammatory cytokines mRNA in liver and epididymal adipose tissue（IL-1β,IL-6 and TNF-α）and intestinal permeability markers in ileum and colon（ZO-1 and Occludin）were measured,in order to investigate the improvement effects of DMY on metabolic endotoxemia and systemic chronic low-grade inflammation resulted from the gut microbiota dysbiosis induced by high-fat diet.Results:DMY at low,medium and high dose could lower the body weight gain,fat mass,weight and lipid content of liver,weight and adipocyte size of epididymal adipose tissue,fasting blood glucose,serum insulin levels,insulin resistance index,AUC of glucose and insulin tolerance tests in high-fat diet-fed mice and high-fat diet induced obesity mice,demonstrating the preventive and therapeutic effects of DMY on high-fat diet induced obesity and insulin resistance.On the contrary to the therapeutic effect of DMY,while the preventive effect was not dose-dependent.After DMY intervention,the levels of serum LPS,proinflammatory cytokines mRNA in liver and epididymal adipose tissue were significantly decreased,while the levels of intestinal permeability markers in ileum and colon were significantly increased.Conclusions:DMY could maintain the intestinal integrity and reduce the level of LPS that pass through the intestinal barrier into the bloodstream,leading to the improvement of metabolic endotoxemia and systemic chronic low-grade inflammation,which resulted in the reducing effects of high-fat diet induced obesity and insulin resistance.The dose-effect relationship of the preventive and therapeutic effects of DMY could provide further experimental evidence for the“hormesis”viewpoint in traditional Chinese medicine.Chapter 4:The composition changes of gut microbiota during DMY intervention.Objectives:Comprehensive analyze the specific modulation effect of DMY on the composition of gut microbiota during the prevention and treatment of obesity and insulin resistance.Methods:Using the 16S rRNA gene sequencing technology with bioinformatics analysis method including OTU analysis and species annotation,sample complexity analysis,comparative PCoA evaluation and STAMP analysis,the changes of diversity and variation of species and relative abundance at different classification levels of gut microbiota after DMY intervention.Results:The results ofαdiversity index analysis and PCoA evaluation showed that the diversity of gut microbiota was altered after DMY intervention.DMY could increase the relative abundance ratio of Bacteroidetes to Firmicutes,of which the effect was dose-dependent.The results of STAMP analysis at different classification levels showed that the relative abundances of some specific species changed by high-fat diet were targeted modulated by DMY.Conclusions:DMY could modulate the gut microbiota diversity during the prevention and treatment of obesity and insulin resistance,and modulate the specific gut microbiota species in association with the development and improvement of obesity and insulin resistance.Chapter 5:In-depth mechanism study of DMY improving obesity and insulin resistance by modulating the composition of gut microbiota.Objectives:Confirm that the modulation effect of DMY on gut microbiota was the mechanism of improving obesity and insulin resistance,and further indentifiy the specific gut microbiota species associated with the development and improvement of obesity and insulin resistance.Methods:At the beginning of establishment of obesity and insulin resistance mouse model induced by high-fat diet,the mice were intervened with antibiotic cocktail before the daily intragastric gavage of DMY,in order to investigate whether the gut microbiota depletion would eliminate the pharmacological effects of DMY.After withdrawal of antibiotic cocktail,the mice were transplanted with the gut microbiota under high-fat diet condition while kept daily intragastric gavage with DMY,in order to investigate whether the pharmacological effects of DMY would recover.On this basis,the composition of gut microbiota was analyzed for the purpose of identifying the specific gut microbiota species in association with the development and improvement of obesity and insulin resistance.Results:The gut microbiota depletion eliminated the pharmacological effects of DMY.After withdrawal of antibiotic cocktail,the transplantation of gut microbiota under high-fat diet condition accelerated the progression of obesity and insulin resistance.After the transplantation of gut microbiota under high-fat diet condition and DMY intervention,the pharmacological effects of DMY were recovered.The composition of gut microbiota analysis indicated that the relative abundance of specific gut microbiota species such as Mucispirillum spp.,Roseburia spp.and Faecalibacterium spp.were signicantly changed.Conclusions:In this chapter,the causal relationship verification method was exploratorily introduced to fully confirm that the modulation effect of DMY on gut microbiota was the mechanism of improving obesity and insulin resistance,which meant that DMY could improve obesity and insulin resistance by targeted modulating the specific gut microbiota species.On this basis,the specific gut microbiota species in association with the development and improvement of obesity and insulin resistance were further identified,including Mucispirillum spp.,Roseburia spp.and Faecalibacterium spp.