| Objectives Through objectiving Shuang-yi hypoglycemic prescription effect on glucose tolerance, glycosylated hemoglobin (HbA1c), blood lipids and fasting insulin levels in type 2 diabetic rats,we can find shuang-yi hypoglycemic prescription's therapeutic effect and discuss it's possible mechanism. Through objectiving the effection on insulin resistance (IR)cells,and discuss Shuang-yi hypoglycemic prescription improve IR's mechanism.Methods Part I: 60 male wistar rats of SPF level, 10 rats were randomly selected as normal control group (NC), normal diet, and the remaining rats were given high fat diet for 4 weeks, two intraperitoneal injection small doses of streptozotocin (STZ). Test of glucose tolerance in rats after a week, we had set fasting blood glucose (FBG)≧ 11.1mmol / L as diabetes mellitus standard. Selecting 40 rats of the FBG in among 11.1 mmol / L -25.0mmol / L ,we randomly had divided 40 rats into 4 groups: Diabetes Mellitus group (DM), Rosiglitazone Maleate positive control group (RM), Shuang-yi hypoglycemic prescription gao dose group ( SG), Shuang-yi hypoglycemic prescription di dose group (SD), n = 10. SG group and SD groups were gavage by 23.43g/kg, 5.86g/kg crude drug dosage of Shuang-yi hypoglycemic prescription, RM group were gavage by 0.29mg/kg dose of Rosiglitazone Maleate, NC group and the DM group were gavage by the same volume pure water. Glucose tolerance was tested on the day before the end of the experiment. Fasting blood HbA1c was tested by tail venous blood on the end date of experiment, then we had collected the abdominal aorta blood , centrifugeding and collecting serum specimens.we had tested blood lipid, fasting serum insulin through serum specimens. Finally we had executed the rats and had left the pancreas,then had fixated for doing HE dyeing. Part II: We had cultured HepG2 cells by the RPMI 1640, in 370C, 5% CO2 conditions.On cells covered the bottom of the bottle , We had digested the cell with trypsin and EDTA. When cells shedding ,We had added FBS into the bottle to stop the reaction.The cell suspension was centrifuged, and we had discard supernatant, and added complete medium.The cells were blowed into cell suspension, count to 105 / ml. The cells were inoculation in 96 pore plates, each pore volume of 200μl, and cultured. When adherent cells accounted for more than 80% of the bottle, we had discarded supernatant, added medium containing different concentrations of insulin, each pore volume 200μl,Meanwhile setting up the normal control group. On insulin effecting 16h, 24h, 36h and 48h, the glucose content of cellular supernatants were tested.At the same time we observed the cellular morphological changes through microscope and through the estimation of the cell activity with MTT method to determine the best IR cell model.With the above experiment process,we had established the best model of IR cells,meanwhile setting up normal control group. After the establishing of IR model group ,we had divided the IR model cells into different doses groups of Shuang-yi hypoglycemic prescription and IR model group. Each group had discarded supernatant and added different concentrations drugs of containing complete medium; meanwhile NC group and DM group joined complete medium.When Drug intervented the cells to 24h in each group, we had tested glucose content of cellular supernatants, discard supernatant, used the Hank's solution to wash one pore , added into MTT solution, added into FBS, then cultured for 4h.We had discarded supernatant, added dimethyl sulfoxide (DMSO) and oscillated to effect 10 minutes, finally tested OD value with a microplate reader.The active ingredients of Shuang-yi hypoglycemic prescription include oleanolic acid, jatrorrhizine, ferulic acid, rhein, loganin, puerarin, daidzin, Astragaloside IV 8 drugs, each drug was divided into 6 concentration.We had doen the experiment in same method.Results The results of glucose metabolism's biochemical indicators show that: SD and RM group's fasting blood glucose and postprandial 0.5h blood glucose and HbA1c values were significantly lower than the DM, and no difference between SD group and RM group; SG group's postprandial 0.5h blood glucose and HbA1c were significantly lower than the DM group, and no difference with the RM group.The results of Lipid metabolism's biochemical indicators show that : SD and RM group's triglyceride (TG) and low density lipoprotein (LDL-C) were significantly lower than the DM model group, and no difference between SD group and RM group.The results of fasting serum insulin show that: SG , SD and RM group's insulin was significantly higher than DM group, and no difference among SG group, SD group and RM group.Spancreas HE dyeing show that: DM group compared with NC group: reduce the number of islets, structural damage, irregular borders, sparse, nuclear reduction; SD and RM group slightly had improved compared with DM group.The cells results show that:①This experiment's the best IR cells model is the concentration of 10-6mol / l insulin incubated 24h.②Concentration of 0.03125 mg/ml of Shuang-yi hypoglycemic prescription solution after intervention IR cell model, can improve IR state.③The results of active ingredient of Shuang-yi hypoglycemic prescription show that: In addition to Astragaloside IV,the 7 kinds of drugs have improved the role of IR and in the 2×10-6 mg/ml—2×10-1 mg/ml range, oleanolic acid ,jatrorrhizine, rhein, and puerarin's the glucose consumption will increase gradually with the increase of the concentration; And ferulic acid, and loganin's the glucose consumption gradually reduced with the increase of concentration; and daidzein is no significant correlated with its concentration ; Astragaloside IV there is no obvious functions in improving IR . No difference in MTT values in each group. Conclusions Shuang-yi hypoglycemic prescription by improving glucose and lipid metabolism, by improving the IR, in a certain degree by restore of the damaged pancreaticβcells, by promoting insulin secretion to play a role of hypoglycemic . |
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