| [Objective]Low dose and multiple intraperitoneal injection of streptozotocin(STZ)induced type 2 diabetes mouse model and spontaneous db/db model and hyperglycemic-induced Ins-1 cells were used as the study objects to determine the effect of huidouba(HDB)on the glucose level of type 2 diabetic mice and explore the mechanism of the improvement of pancreatic beta cell function.[Methods]Diabetes mellitus was induced using STZ as described previously.After mice were fasted for at least 8 h in the morning,stable diabetes was induced by successive intraperitoneal injections of STZ(50 mg/kg body weight)dissolved in 0.1 M sodium citrate buffer(pH 4.5)to the mice once a day,5 d in a row.The control group rats received only citrate buffer once a day,5 d in a row.The mice then received a standard diet and water ad libitum for 1 week.One week after STZ injection,fed plasma glucose and fasting plasma glucose levels were measured using a glucometer.Only mice with fed blood glucose levels over 16.7 mmoL/L were considered as diabetic mice and used for the experiment.The mice used in the experiment were divided into one group of the normal control group(NCG)and six groups of diabetic mice(n=10 in each group).There were diabetic group(DG),high dosage of HDB group(HDG,48 g/kg),medium dosage of HDB group(MDG,24 g/kg),low dosage of HDB group(LDG,12 g/kg),metformin group(MG,0.2 g/kg)and acarbose group(AG,0.04 g/kg).Doses were administered orally for 35 d.The mice in the normal control group and diabetic group were given equal volumes of water.Food intake,water intake,fasting plasma glucose level,and the body weights of all animals were monitored throughout the experiment.The db/db mice were divided into five groups(n=10/group)until the age of 8 weeks when fed blood glucose levels were over 16.7 mmol/L and fasting plasma glucose levels reached 11.1 mmol/L.The wild-type mice(n=10),i.e.,the normal control group(Control),were used for comparison with the diabetic groups.The db/db mice were grouped into the diabetic group(Model),HDB treatment group(HDB,48 g/kg,the dosage of HDB extract was determined according to the preliminary experiment),metformin(Met,0.2 g/kg).Body weight and fasting plasma glucose level were measured weekly during the administration period.Serum lipid levels were detected by biochemical kit.Serum insulin content and proinsulin content were detected by Elisa.And the expressions of GLUT2,NKX6.1 and MafA and other pancreatic islet cell related genes and proteins were detected by real-time PCR and WB,respectively.LC-MS was used to detect 70%ethanol extraction of HDB,and Compound Discovery software was used to extract and pretreat the detection data,including retention time,molecular weight,peak strength and other information.Based on the molecular weight,the full spectrum identification was carried out based on the network database Metlin.The identified substances mainly include amino acids,flavonoids,terpenoids,organic acids and so on.According to the results of LC-MS analysis,the standard substance with high ionic response strength was selected for verification and analysis.By comparing the retention time of HDB alcohol extraction with the standard substance and its pyrolysis characteristic sub-ions,the identification and analysis preliminarily determined that HDB contained P-Coumaric acid,Ferulic acid,Luteolin and Quercetin.INS-1 cells are commonly used to test insulin secretion.INS-1 cells were continuously incubated in high glucose medium(containing 33.3mmoL/L glucose)in vitro for 72h to induce high glucose injured cell model.The high glucose injured cells were divided into model group,HDB high,medium and low administration group,quercetin,luteolin group,p-coumaric acid group,ferulic acid intervention group and positive control metformin group.After the intervention,cell viability,glucose-stimulated insulin secretion,real-time PCR and western blot were used to detect the changes of GLUT2,NKX6.1 and MAFA,respectively.[Results]1.The alcohol extraction of HDB could improve the glucose tolerance injury and reduce the blood glucose levels in STZ-induced diabetic mice(1)STZ-induced diabetic mice were effectively relieved of "three more and one less" symptomsOn the second week of administration;Water and food intake were significantly decreased in the high,medium and low groups compared with model group.(2)It can effectively reduce the blood glucose levels in STZ diabetic miceOn the seventh day of administration,the STZ-induced diabetic mice in the group with HDG could significantly reduce the random blood glucose level.The effect of HDG on STZ-induced random blood glucose level in diabetic mice lasted until the end of administration.In addition,the fasting blood glucose level in STZ-induced diabetic mice in the high dose group was also decreased at 28 days of administration.(3)HDB could enhance the glucose tolerance and increase insulin sensitivity in diabetic mice induced by STZThere was no improvement in oral starch tolerance and sucrose tolerance.However,the glucose tolerance in STZ-induced diabetic mice was significantly improved in each group in the high-dose group;middle dose and low dose groups.The insulin sensitivity in STZ diabetic mice was also significantly enhanced in the HDG.2.The HDB promoted the insulin secretion to reduce the blood glucose levels of in db/db mice(1)Effect of HDB on blood glucose level in db/db miceAt the 21 st day of administration,HDB could significantly reduce the fasting glucose level,and the effect lasted until the end of administration.HDB could improve the glucose tolerance in db/db mice.Insulin sensitivity was increased in db/db mice,but there was no statistical differences.There was no effect on the body weight in db/db mice.(3)HDB could improve the dyslipidemia in db/db mice.HDB increased the high-density lipoprotein level,decreased the low-density lipoprotein level and triglyceride level in db/db mice,but had no significant effect on the total cholesterol level.(4)HDB promoted the insulin secretion in db/db miceHDB could increase the serum insulin content,reduce the serum proinsulin content,and reduce the ratio of serum proinsulin to insulin.(5)HDB was used to repair the histopathological changes of the pancreas in db/db miceHistopathological changes of the pancreas in db/db mice were observed by HE staining.In the HDB group,the shape of the islets was regular and the boundary was clear,and the vacuolar lesions in the islets were reduced and the inflammatory infiltration was alleviated.Immunohistochemical staining was performed to observe the secretion of somatostatin and pancreatic polypeptide by islet ? cells and PP cells.Compared with the model group,HDB reduced the positive expression of somatostatin and pancreatic polypeptide(P<0.05),? cells and PP cells tended to distribute around the islets.Immunofluorescence staining was performed to observe the secretion of glucagon and insulin by ? cells and ?cells.The HDB enhanced the fluorescence intensity of insulin and glucagon and increased the number of insulin positive expressions.3.there were p-coumaric acid,ferulic acid,luteolin and quercetin in 70%ethanol extract of HDBLC-MS identified and analysised 70%ethanol extraction of huidouba.By comparing the retention time and pyrolysis characteristic ion of HDB with the standards,it was preliminarily determined that 70%ethanol extraction of huidouba contained p-coumaric acid,ferulic acid,luteolin and quercetin.4.HDB and its components can repair the damage of INS-1 cells induced by high glucose(1)HDB and its components can increase the activity of INS-1 cells induced by high glucoseThe cell viability of INS-1 cells cultured in high glucose medium for 72h decreased significantly.Compared with the cells cultured in normal medium,the cell viability of INS-1 cells damaged by high glucose decreased significantly.The cell viability of HDB high-dose group,medium-dose group,low-dose group,quercetin group,ferulic acid group and metformin group was significantly increased compared with that of high-glucose injury model group.The cell viability of luteolin and coumaric acid intervention group was increased compared with model group,but there was no statistical significance.(2)HDB and its components can inhibit the apoptosis of INS-1 cells induced by high glucoseThe Caspase-3 and Caspase-8 activities of INS-1 cells were significantly increased after 72 hours of continuous culture in high glucose medium.The activities of Caspase-3 and Caspase-8 in the high dose group of HDB were significantly lower than those in the high glucose induced model group,the activities of Caspase-8 in the quercetin group were lower,and the activities of Caspase-8 in the ferulic acid group were lower.(3)HDB and its components can promote basal insulin secretion and glucose-stimulated insulin secretion in INS-1 cells.[Conclusion]1.There were typical symptoms of "three more and one less" in STZ induced diabetic mice.The blood glucose of the mice increased rapidly after intraperitoneal injection of 50 mg/kg STZ for 5 days,reaching the diagnostic criteria of blood glucose level of type 2 diabetes:random blood glucose? 16.7 mmoL/L;Fasting blood glucose?11.1 mmoL/L.C57BL/KSJ-db/db spontaneous diabetic mouse model is induced by point mutation of leptin receptor,leading to reduced sensitivity of insulin receptor,hyperlipidemia and obesity,and widely used in the study of the pathogenesis of type 2 diabetes.2.HDB can reduce the blood glucose levels of STZ-induced diabetic mice and db/db mice,improve insulin sensitivity of the two kinds of diabetic.mice,improve lipid disorder in obese mice,promote insulin secretion of db/db mice,and repair pathological damage of pancreas of diabetic mice.3.The hypoglycemic action mechanism of HDB may be to maintain the identity of pancreatic ? cells and the characteristics of insulin secretion by regulating the expression of islet ? cell-related transcription factors.4.Incubation of INS-1 cells with high glucose for 72 h inhibited the activity and insulin secretion of INS-1 cells,promoted cell apoptosis,and inhibited GSIS.The alcohol extraction of HDB and quercetin could significantly inhibit the damage of ?-cells induced by high glucose,inhibit the apoptosis of ?-cells induced by high glucose,improve the cell viability,and promote GSIS. |
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