| Objectives:Diabetes mellitus(DM)is a chronic disease that occurs when the body is completely unable to secrete insulin,or secrete enough insulin,or effectively use insulin,resulting in elevated glucose levels in the blood.Type 2 diabetes is the most common type of DM,which accounts for approximately 90%of all cases of diabetes.In type 2 diabetes,insulin resistance,hyperglycemia,and hyperlipidemia are generally present.Since type 2 diabetes patients are generally caused by obesity and their lipid metabolism is abnormal in the body,in recent years,more and more people are paying attention to finding new targets for treating type 2 diabetes with bile acids.Bile acids can regulate lipid and glucose metabolism by affecting the FXR and TGR5 signaling pathways,which lower,blood sugar by stimulating glucagon-like peptide-1(GLP-1)to reduce insulin.The bile acid-related synthetase Cyp8bl may be an effective target for the treatment of type 2 diabetes,but the exact mechanism is not clear:After weight-loss surgery in patients with type 2 diabetes,blood glucose levels,insulin resistance,and body weight were improved,and serum bile acid levels increased after surgery,indicating that bile acids can indeed be used as therapeutic targets for type 2 diabetes.At present,the first-line drugs for clinical use in type 2 diabetes have certain adverse reactions or side effects,and some can cause certain damage to the liver.They are not recommended for patients with impaired liver function.However,it has been reported in the literature that berberine was used in the treatment of diabetic patients,which can reduce the body weight of the patient and caused no damage to the liver.In addition to hypoglycemic effect,berberine also reduces triglyceride,cholesterol and low-density lipoprotein levels.On the other hand,bile acids play an important role in fat metabolism.Therefore this study investigated the changes of 24 major bile acids in the liver,gallbladder,ileum,colon,serum,and feces in the normal control mice,type 2 diabetes mellitus and berberine-treated mice.The 24 major bile acids are CA,CDCA,UDCA,DCA,HDCA,LCA,HDCA,MCA,α-MCA,β-MCA,ω-MCA,T-CA,T-CDCA,T-UDCA,T-HDCA,T-DCA,T-LCA,T-α-MCA,T-β-MCA,G-CA,G-UDCA,G-CDCA,G-DCA and G-LCA.UHPLC-MS/MS was used to quantify bile acids,and it was intended to elucidate the mechanism underlying berberine’s effect on the reduction of reduces triglyceride,total cholesterol and low-density lipoprotein levels in type 2 diabetes mellitus mice through bile acids metabolism.Methods:1.Berberine administration experimentSix-week-old db/m mice were used as normal controls.db/db mice were randomly divided into four groups:type 2 diabetes model group(T2DM group,n=6)and berberine low dose group(150 mg/kg BBR group,n=6)and berberine high dose group(300 mg/kg BBR group,n=6),all groups have male and female groups,respectively.The fasting blood glucose before the administration was measured,the pre-dose serum sample was collected,and then the administration was started,and the administration dose was given once a day,and the control group and the model group were intragastrically administered with the same volume of physiological saline per day.During the treatment,the body weight of the mice was monitored every week,and the fasting blood glucose levels were monitored every month until the end of the experiment,and biological samples of each mouse were collected.2.Liver histopathologyAfter the treatment of each group of mice,two small portions of fresh liver tissue were taken in tissue fixative,then paraffin sectioning technique was used for HE staining,and cryosection technique was used for oil red staining to investigate berberine’s effect on mouse liver tissue.3.Detection of tissue,feces and serum bile acid levelsUsing the UHPLC-MS/MS method established in our laboratory,24 major bile acids in the liver,gallbladder,serum,ileum,colon,feces and urine of each group were detected.4.Detection of protein expression levels of bile acid nuclear receptors,synthetases and transportersWestern blotting was used to detect the expression levels of bile acid nuclear receptors,synthetase and transporter proteins in the bile acid enterohepatic circulation in each group.Results:1.Berberine slowed the increase in body weight and fasting blood glucose in type 2 diabetes mellitus miceCompared with the normal control group,the weight gain and fasting blood glucose of the type 2 diabetes model group and the treatment group were significantly different,while the berberine treatment groups were compared with the type 2 diabetes model group;the increase in body weight and fasting blood glucose was significantly slowed down.2.Berberine improved liver function in type 2 diabetes mellitus miceBerberine could reduce the liver index of type 2 diabetes mellitus mice,and berberine could improve liver vacuole lesions and reduce lipid droplet formation in type 2 diabetes mellitus mice.In the measurement of total cholesterol and triglycerides,berberine was shown to reduce serum total cholesterol and triglyceride levels in type 2 diabetes mellitus mice.3.Results of various tissues,feces and serum bile acid levels3.1.Results of feces and urine bile acid levelsIn feces and urine,type 2 diabetes mellitus mice had a reduced total bile acid content,which,after treatment with berberine,significantly increased their fecal total bile acid content.3.2.Results of serum bile acid levelsIn type 2 diabetes mellitus mice,the proportion of taurine-bound bile acid in serum was significantly increased compared with the normal control group,and after treatment with berberine,the proportion of taurine-bound bile acid was reduced.3.3.Results of liver bile acid levelsIn the normal control group,the proportion of taurine-bound bile acid in the liver was significantly increased in the type 2 diabetes mellitus mice,and the results were reflected in both male and female mice.After treatment with berberine,the proportion of taurine-bound bile acids could be reduced.The total bile acid content in the liver of type 2 diabetes mellitus mice was significantly reduced.After treatment with berberine,the total bile acid content in type 2 diabetes mellitus mice was increased.3.4.Results of ileum and colon bile acid levelsCompared with the normal control group,the proportion of taurine-bound bile acids in the ileum tissue samples was significantly increased in the type 2 diabetes mellitus mice.After treatment with berberine,the ratio of taurine-bound bile acids in ileum could be reduced,but the effect was not obvious.In the colon,low-dose berberine treatment significantly reduced the proportion of taurine-bound bile acids in type 2 diabetes mellitus mice.In ileum and colon,type 2 diabetes mellitus mice had an increased total bile acid content,but treatment with berberine reduced the total bile acid content in the ileum and colon of type 2 diabetes mellitus mice.3.5.Results of gallbladder bile acid levelsIn the gallbladder samples,the bile acid content of each group was dominated by taurine-bound bile acids.The total bile acid content of type 2 diabetes mellitus mice was significantly increased compared with normal control mice,and the administration of berberine reduced the total bile acid content in the bile samples of type 2 diabetes mellitus mice.3.6.Berberine up-regulated protein expression levels of hepatic bile acid synthaseAfter treatment with berberine,it could significantly increase protein expression levels of bile acid synthase,Cyp7a1,Cyp8b1 and Cyp27al in the liver of type 2 diabetes mellitus mice.Conclusion:Bile acids metabolism was disrupted in type 2 diabetic mice,but after treatment with berberine,it can slow the increase of body weight and fasting blood glucose in type 2 diabetes mellitus mice,and can reduce liver fat accumulation and improve liver function.In each tissue site,it can reduce the proportion of taurine-bound bile acids content,increase the total bile acid content of the liver,reduce the total bile acid content of the ileum and colon and increase the total bile acid content in the feces.The protein expression of synthetase indicates that berberine can increase bile acid synthesis in the liver of type 2 diabetes mellitus mice,reduce the reabsorption of bile acids in the ileum and colon,and increase the excretion of bile acids from feces and urine. |
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