Study Of Hypoglycemic Activity And The Mechanism Of Extracts From Edgeworthia Gardneri(Wall.) Meissn

Type 2 diabetes mellitus(T2DM)has become one of the most prevalent chronic diseases worldwide.Insulin resistance(IR)is a driving factor in the development and progression of T2 DM.Therefore,it is urgent to find effective drugs to treat T2 DM by improving IR.The flower of Edgeworthia gardneri(Wall.)Meissn(E.gardneri)is a traditional medicine which has good therapeutic effecacy on hypoglycemic and hypolipidemic.It has been reported that the flower of E.gardneri can effectively treat T2 DM by inhibiting ?-glucosidase activity and activating PPAR?/?.Based on the previous work and combined with compositions of E.gardneri,we suggest that it may improve the IR to achieve hypoglycemic effect in order to treat T2 DM.Therefore,in this study,different solvent extracts that can improve IR were isolated from the flower of E.gardneri and the study of hypoglycemic mechanism was carried out to provide basis for the treatment of T2 DM.The main results are as follows:(1)To establish the IR cell model of myotubes and evaluate the hypoglycemic activity of different solvent extracts from the flower of E.gardneri in vitro.First,five different differentiation media were used to induce C2C12 myoblasts into myotubes.Through detecting the morphology of cells,creatine kinase activity,mRNA and protein expression during differentiation,the best differentiation condition was cultured C2C12 cells with the high glucose DMEM medium contain 2% horse serum for 5-7 days.Then,the myotubes were cultured with 0.75 mM palmitic acid for 16 h to establish the IR cell model and evaluated the hypoglycemic activity of different solvent extracts from the flower of E.gardneri.The results showed that the extract of n-hexane from the flower of E.gardneri(named EGH in following study)had a good hypoglycemic activity.At the concentration of 25-200 ?g/m L,EGH could dose-dependently increase the consumption and uptake of glucose in C2C12/IR cells.(2)To evaluate the hypoglycemic activity of EGH with T2 DM mice models.One of them were C57BL/6J mice which were induced though high-fat diet combined with low-dose streptozotocin,another were db/db mice which were spontaneously formed.The results showed that after 4 weeks treatment of EGH(40,80 and 160 mg/kg/day),fasting blood glucose,oral glucose tolerance and glycosylated hemoglobin were decreased in a dose-dependent manner,indicating that EGH had good hypoglycemic activity.The results of HE staining and detection of IR-related indicators(fasting serum insulin,insulin resistance index,?-cells function index,insulin activity index)showed that EGH could repair damaged islet tissue to promote insulin release and improve muscle tissue sensitivity.EGH also decreased free fatty acids,triglycerides and total cholesterol,which indicated that EGH could regulate lipid metabolism.Also,EGH could promote the phosphorylation of insulin receptors(IRs)and insulin receptor substrate-1(IRS-1)in skeletal muscle,and increase the expression of glucose transporter 4(Glut4).(3)To screen the hypoglycemic active components in EGH and explore mechanism.After the preliminary investigation of the hypoglycemic mechanism of EGH,five components(Fr.1-Fr.5)were isolated by gel chromatography on silica gel,and the hypoglycemic activity of Fr.1-Fr.5 was evaluated by C2C12/IR cells.In the range concentration of EGH(12.5-100 ?g/m L),Fr.1,Fr.3 and Fr.5 could increase the consumption and uptake of glucose in C2C12/IR cells,with Fr.1 being the best and then Fr.3.In further study of Fr.1 hypoglycemic mechanism,we found that Fr.1 could increase p-IRs and p-IRS-1 activate intracellular phosphatidylinositol kinase(PI3K),downstream negative regulatory tumor suppressor gene protein(PTEN)phosphorylation and promote phosphorylated protein kinase 1(PDK1)phosphorylation,further promoting the downstream protein kinase B(AKT)and glycogen synthase kinase 3?(GSK 3?)phosphorylation.Fr.1 also activated the AMPK signaling pathway by promoting phosphorylation of AMP-activated protein kinase ?(p-AMPK?)and acetyl Co A carboxylase(p-ACC).In summary,Fr.1 could increase the expression of Glut4 by promoting PI3K/AKT and AMPK signaling pathways,thus promote the uptake of glucose to improve IR to achieve treatment of T2 DM.