Chlorogenic Acid Alleviates Type 2 Diabetes Mellitus Via Microbiota In Db/db Mice

The Chlorogenic acid is the an effective ingredient of traditional Chinese medicine such as honeysuckle,eucommia and oriental wormwood,which is also rich in tobacco.Studies have shown that Chlorogenic acid not only has beneficial effects on lowering glucose and lipids concentration in serum and improving insulin sensitivity,but also significantly altering the composition of animal intestinal microbial population.In order to explore the effect and mechanism,whether intestinal microbes play the key role of chlorogenic acid in improving type 2 diabetes,this thesis study effects of chlorogenic acid on biochemical index of db/db mice,which used the db/db mice as models and the db/m mice as control.The effect of chlorogenic acid on glucose tolerance and insulin sensitivity in db/db mouse were studied,which tolerance test of oral glucose and insulin with empty abdominal injection was be used.The effect of chlorogenic acid on the cellular morphological structure of islet and liver and content of liver fat were studied by staining of HE and oil-red O.The effect of chlorogenic acid on pancreatic cell apoptosis,hepatic lipid metabolism,mitochondrial function and expression of genes associated with inflammation and oxidative stress were studied by using fluorescence quantitative PCR technology.The effect of chlorogenic acid on PI3K/AKT pathway protein was studied by applying protein immunoprinting method.The effect of chlorogenic acid on intestinal microbial composition and short-chain fatty acid content were studied by using 16 S r RNA microbiological sequencing technology and gas chromatography.The important role of chlorogenic acid on improving intestinal microorganisms of type 2diabetes was studied by applying antibiotics and fecal bacteria transplantation techniques.The main research results obtained are as follows:1.The chlorogenic acid significantly deceased body weight,adipose tissue and liver weigh of db / db diabetic mouse,and reduced the glucose concentration in serum,as well as improved glucose tolerance and insulin sensitivity.The chlorogenic acid significantly reduced triglyceride content in serum,lipid accumulation in liver and improved hepatic lipid metabolism and mitochondrial function.It also significantly improved the cellular morphological structure of islet and liver,alleviated oxidative stress and inflammatory responses,and improved the expression of PI3 K and AKT phosphorylation proteins.2.The chlorogenic acid significantly increased the intestinal microbial α-diversity index,but the composition and proportion of mouse intestinal microbes in different levels(phylum,class,order,family,genus and species)in the chlorogenic acid-treated mouse were the same as the db/m mouse,while it was significantly different between no the chlorogenic acid intervention group and other groups.The chlorogenic acid significantly increased butyric acid content in the mouse caecum,excepted acetic acid,propionic acid,valeric acid.In addition,the beneficial effects of chlorogenic acid above mentioned on diabetic db/db mouse were similar to metformin.3.After antibiotics inhibit the intestinal flora of db/db mouse,it was found that chlorogenic acid has no significant impact on body weight,adipose tissue,liver weight,glucose and triglyceride concentration in serum,glucose tolerance,insulin sensitivity,triglyceride content in blood and fat deposits in liver of db/ / db mouse by pretreated with antibiotics.There was no significant effect on hepatic lipid metabolism,mitochondrial function,oxidative stress,inflammatory response,cellular morphological structure of islet and liver,and the expression of PI3 K and AKT phosphorylation proteins in the db/db mouse.4.When the db/db mouse with antibiotics inhibiting intestinal flora were transplanted the feces which collected from db/db mouse and blended in chlorogenic acid,it was found that the mouse feces could significantly reduce body weight,fat tissue and liver weight,as well as glucose and triglyceride concentration in serum.They had prominently improved glucose tolerance,insulin sensitivity,lipid accumulation in serum and fat deposits in liver of mouse,and improved hepatic lipid metabolism,mitochondrial function,oxidative status,inflammatory responses and cellular morphological structure of liver and islet.It can also had significantly increased the expression of phosphorylated proteins of PI3 K and AKT in downstream of insulin signaling pathway,as well as the content of butyric acid in cecum.In addition,it could also improve microbial abundance in the level of phylum,class,order,family,genus and species respectively on Proteobacteria,Verrucomicrobiae,Verrucomicrobiales,Akkemansiaceae,Bacteroidaceae,Rikenellaceae,Akkemansia,Bacteroides,Akkemansia＿muciniphila,Bacteroides＿caecimur is and so on.In conclusion,the chlorogenic acid could significantly improve the glucose and lipid metabolism,mitochondrial function,oxidative status,inflammatory responses,oxidative status,cellular morphological structure of liver and islet of mouse.It also improved the expression of phosphorylated proteins of PI3 K and AKT,promoted the recovery of intestinal microbial steady-state and improved the butyric acid content in cecum of mouse.Experiments of antibiotic treatment and fecal bacteria transplantation showed that intestinal microorganisms may play a key role in chlorogenic acid alleviating type 2 diabetes.This results suggested that fecal microbial transplantation could be used as a potential therapeutic method for the treatment of type 2 diabetes.