Mechanism Of Vaccarin Improving Glucose And Lipid Metabolism Disorder In Type 2 Diabetes Through Intestinal Flora-bile Acid Axis

Objective: Type 2 diabetes mellitus(T2DM)is a metabolic disorder that seriously endangers human life and health,and its prevalence is increasing worldwide.Currently,the mechanisms of T2 DM development are not fully elucidated.Numerous studies have shown that intestinal flora and bile acids,a metabolite of flora,play an important role in the development of T2 DM.Vaccarin(VAC)is a flavonoid phytochemical extracted from the seeds of Vaccinium macrocarpon,which has various biological functions.Previous studies have found that VAC has the effect of improving the disorder of glucolipid metabolism in T2 DM mice,but its specific mechanism needs to be further elaborated.The aim of this study was to investigate the effect of VAC on the disorder of glucolipid metabolism in T2 DM through the intestinal flora-bile acid axis and its specific mechanism.Methods: The study was divided into four parts as follows:Part Ⅰ: To investigate the effects of VAC on disorders of glucolipid metabolism,intestinal flora and the flora metabolite bile acids in T2 DM mice.A high fat diet(HFD)combined with intraperitoneal injection of streptozotocin(STZ)was used to construct a T2 DM mouse model,and the experiment was divided into CON group,T2 DM model group and T2DM+VAC group.Fasting blood glucose,insulin sensitivity and glucose tolerance were measured by tail vein blood collection;serum biochemical parameters and liver glucose and lipid metabolism parameters were measured by using kits and real-time quantitative PCR(q RT-PCR);intestinal flora composition of mice in each group was detected by 16 S r RNA technology and q RT-PCR;The changes of bile acid profile were determined by ultra performance liquid chromatography-mass spectrometry(UPLC-MS).Part Ⅱ: Fecal microbiota transplantation(FMT)and antibiotic-depleted flora experiments were performed to investigate the role of intestinal flora in the anti-diabetic process of VAC.In the FMT experiments,CON,T2 DM and T2DM+VAC mice were used as donors and their feces were transplanted to the same physiological state of the recipient mice.In the antibiotic experiment,the T2 DM mice were supplemented with 200 μL of antibiotic solution(neomycin1 g/L,ampicillin 1 g/L,vancomycin 0.5 g/L,metronidazole 1 g/L,sterile saline)daily for one week,and the T2 DM mice were depleted of their flora before the VAC intervention was given,and the therapeutic effect was compared with that of the T2DM+VAC group.The changes of intestinal flora and glycolipid metabolism related indexes in mice were measured by the same method as in the first part.Part Ⅲ: Screening and investigation of bile acids that play a beneficial role in the improvement of glycolipid metabolism by VAC in T2 DM mice and their mechanisms.Differential bile acids that are closely related to the body’s glycolipid metabolism were screened by Spearman correlation analysis.Next,we used oleic acid combined with palmitic acid to induce insulin resistance model in human hepatocellular carcinoma cells(Hep G2)to observe the effect of the screened differential bile acids on the disorder of glycolipid metabolism in Hep G2 cells;q RT-PCR,protein immunoblotting and transient transfection experiments were used to investigate the specific signaling pathways of bile acids affecting the body’s glycolipid metabolism.T2 DM mice were constructed and intervened with bile acids to verify the effects and mechanisms of differential bile acids on glycolipid metabolism in T2 DM mice.Part Ⅳ: To investigate the beneficial bacteria and their effects on regulating differential bile acid metabolism and thus improving the disorders of glucolipid metabolism in T2 DM mice.By Spearman’s correlation analysis,we screened out the beneficial bacteria that were significantly correlated with the differential bile acid content and glucolipid metabolism index in the third part,and their content could be significantly increased by VAC intervention.After constructing a T2 DM mouse model,the screened beneficial strains were supplemented orally.The same method was used to observe the glucolipid metabolism level and bile acid metabolism in T2 DM mice after supplementation with beneficial bacteria.Results: Part Ⅰ: VAC intervention effectively lowered blood glucose,improved glucose tolerance,reduced insulin resistance and lowered lipids in mice with T2 DM compared to mice with T2 DM model.Meanwhile,the intestinal tissues of T2 DM mice showed obvious pathological changes and dysregulation of intestinal flora homeostasis.VAC could regulate the composition and structure of intestinal flora in T2 DM mice,increasing the content of Bacteroidota and decreasing the content of Firmicutes,and multivariate statistical analysis of intestinal flora showed that intestinal flora has an important influence on the physiology of the organism.The multivariate statistical analysis of intestinal flora showed that intestinal flora has an important influence on the physiology and pathology of the organism,and intestinal flora is closely related to the level of glucolipid metabolism.In T2 DM mice,total bile acids were increased in serum and liver compared with normal mice,and VAC intervention reduced total bile acid levels and regulated bile acid homeostasis in mice.Part Ⅱ: Flora transplantation experiments revealed that reconstituting the intestinal flora of mice could affect the glucose,lipid and liver glucolipid metabolism levels of the organism,and the glucolipid metabolism of mice transplanted with flora from the T2DM+VAC group was superior to that of mice transplanted with flora from the T2 DM group.On the other hand,the effect of VAC in treating T2 DM was diminished after depleting the mouse colonies with antibiotics.This suggests that VAC exerts its effect on treating disorders of glucolipid metabolism in T2 DM mice partly through the intestinal flora.Part Ⅲ: Spearman’s correlation analysis showed that the levels of Cholic acid(CA)and Chenodeoxycholic acid(CDCA)were negatively correlated with blood glucose,blood lipids and liver glucolipid metabolism indexes in mice,suggesting that CA and CDCA may be the key bacterial metabolites regulating the body’s glucolipid metabolism.Supplementation of CA and CDCA can effectively improve the disorder of glucolipid metabolism in Hep G2 cells;meanwhile,CA and CDCA can effectively alleviate insulin resistance and improve liver function in T2 DM mice,which has good hypoglycemic effect.The mechanism is related to the activation of TGR5/c AMP/AMPK expression in the liver.Part Ⅳ: Spearman correlation analysis screened that Bacteroides uniformis was closely associated with CA,CDCA and glucolipid metabolic indexes in mice,suggesting that it may be a target for VAC treatment of T2 DM as a beneficial bacterium.Supplementation of Bacteroides uniformis could effectively reduce blood glucose and lipid levels in T2 DM mice,increase insulin sensitivity and regulate glucolipid metabolism disorders.This effect was associated with the fact that Bacteroides uniformis intervention increased bile salt hydrolase(BSH)levels in the intestine of T2 DM mice,which in turn increased CA and CDCA levels and upregulated the expression of the subsequent bile acid pathway TGR5/c AMP/AMPK.Conclusion: In conclusion,VAC can alleviate the dysregulation of intestinal flora homeostasis in T2 DM mice,increase the level of Bacteroides uniformis in the intestine,increase the BSH content,thus increasing the level of intestinal flora metabolites CA and CDCA in the hepatic and intestinal circulation,activate the expression of TGR5/c AMP/AMPK in the liver,and finally improve the glucolipid metabolic disorders and lower blood glucose.This study suggests that VAC can be used as a beneficial natural antidiabetic nutrient,and further enriches the theory that the intestinal flora-bile acid axis affects the development of T2 DM,providing new targets and new ideas for the future clinical prevention and treatment of T2 DM.