| Background&Objective:Diabetes, ranking just after cancer and cardiovascular and cerebrovascular diseases, is one of the most harmful diseases to human on health and economies.90-95% of the diabetic patients are belongs to NIDDM, and 60-90% of the NIDDM patients are overweight. Therefore, to establish an animal model whose pathophysiologic changes are similar to human diabetes, may provide better understanding and treatments on diabetes.Combination of high-fat diet and STZ is commonly used in model establishment of typeⅡdiabetes. Generally, high-fat diet (30%of fat) feeding and a small amount of STZ (200mg/kg) injection in mice has been considered to be a simple, economical and time saving method to establish model of typeⅡdiabetes. Such model can provide good evaluation of potential medicine that may treat typeⅡdiabetes and make the pathomechanism and related complications of type II diabetes more clear so that further study can be continued. However, we found that there are still some disadvantages like unsteadiness, bad evaluation of some drug, invalid evaluation of insulin secretagoguesn in this model, which might cause omission when screening new drugs that have potential value in treating typeⅡdiabetes. Therefore, further optimization of this model is obviously necessary.There are three main factors of this model including dose of single dose, time of the first injection and injected frequency according to many documents and previous study. In order to find out the best combination, three factors and three levels orthogonal test is adopted and three factors and three levels are:single does (75mg/kg,85mg/kg,100mg/kg), time of the first injection (the first,the second,the third week), injected frequency (1,2,3 times), respectively. Find out the best combination through orthogonal test according to the orthogonal layout. Finally, examine the stability and repeatability of the model and confirm the area of application. Aim:To optimize a rodent model of type II diabetes that would replicate the metabolic characteristics of the human syndrome and the suitable for pharmaceutical research.Methods:1.The three factors and three levels orthogonal test:In order to find out the best combination, three factors and three levels orthogonal test is adopted and three factors and three levels are:time of the first STZ injection (a1=the first week,a2=the second week,a3=the third week), single does (b1=75mg/kg,b2=85mg/kg,b3=100mg/kg), injected frequency (c1=1 time,c2=2 times,c3=3times), respectively. According to the orthogonal layout, combinations are ranked as followed:a1b1c1, a1b2c2,a1b3c3(c2), a2b1c2, a2b2C3(c2), a2b3c1, a3b1c3,a3b2c1, a3b3c2, Inadequate combinations are rejected on the basis of the previous documents and replaced by the level in the brackets. There-week-old weaning C57BL/6J mice were fed with high-fat diet containing 30% of fat then divided into 9 groups (8 animals per group). Weight and blood glucose were recorded every week. Fasting blood glucose was measured before the STZ injection. The method of fasting blood glucose testing is as followed:mice were fasted but free access to water for 12 hours (08:00pm-08:00am). Blood samples were collected by cutting the tail tip and tested through blood glucose meters (type: steadily SureStep, supplied by Johnson & Johnson of the United States). Animals were fed with high-fat diet sequentially after STZ injection. The changes of body weight, food intake and blood glucose were measured every week. The mice were decapitated five weeks after STZ injection, and the pancreata were taken for pathological section.2. Oral glucose tolerance test Overnight-fasted (15 hours) mice were given a glucose solution orally (20% glucose if fasting glucose<150mg/dl or 10% glucose) and blood samples were collected from tail vein at 30,60 and 120 minutes after the glucose administration. Blood glucose curve diagram was done. Deal with the data with repeated measurement and compare the diversity between groups, a=0.05)3. Plasma chemistry:Plasma free fatty acids, insulin level were tested with corresponding testing kits. All detecting procedures were done according to the kit instructions.4. Histopathological detection:Pancreata of the mice were collected with scissors and scalpel after decapitation. After fixed in 4% of paraformaldehyde for more than 24h, the pancreatic tissue was dehydrated by gradient alcohol, transparentized by dimethylbenzene, embedded by wax and kept overnight at 4℃. The samples were cut into slices of 4um then affixed to the slide on the antidepinning siliconization. The dried samples were stained with HE. Pathological changes were observed in the optical microscope. Evaluate the putrescence,apoptosis and denaturation of the pancreata.5. Investigation of stability and reproducibility of the optimize model We used different batches of mice and observed for more than 8 weeks to investigate the stability and reproducibility of the best combination.24 C57BL/6J mice were divided into three groups (8 per group):model group, control group and high-fat diet group.6. Pharmacodynamic evaluation of the optimized model:To determine Pharmacological effects of the optimized Fat-fed/STZ mice model, we tested three kinds of drugs widely used in type 2 diabetes.100 C57BL/6J mice were used in the test and 80 were fed with high-fat diet,20 low-fat diet.70 high-fat-diet mice and 10 low-fat-diet mice were injected with STZ according to the results of orthogonal experiment. Choose 64 animals from high-fat-diet/STZ mice and then divided them into 8 groups:normal control, negative control and medication administration group. Three kinds of drugs were choosen for testing:metformin(200mg/kg/d,500mg/kg/d), glimepiride(0.1mg/kg/d,0.3mg/kg/d) and pioglitazone(10mg/kg/d,30mg/kg/d). Mice were randomized by computer 1 day before administration according to blood glucose concentration and body weight. Intragastric administration were executed 1 time per day (10mL/kg) and continued for 21 days.Results:1. The result indicates that injection of streptozotocin(85mg/kg) twice every five days at the third week is the best for establishing rodent type II diabetes model. Biopsy also showed appropriate destruction of islet.2. The optimized model is stable and reproducible. The success ratio of model establishment is 90%. The blood glucose concentration was stable and can sustain until eight weeks after STZ injection.3. Fat-fed/STZ mice is sensitive to metformin and pioglitazone. Glimeperide also has hypoglycemic effect when administrating dose is up to 0.3 mg/kg.4. Test results of blood triglyceride and free fatty acid shows that these three kinds of drugs used in the model could well ameliorate the high triglycerides and high-fatty acids, particularly metformin and pioglitazone high-dose group. Glimepiride low-dose group is about 16%lower than the negative control group.Conclusions:1. The rodent model of typeⅡdiabetes is successfully optimized (pathology is more similar to type 2 diabetes of human) and the time of inducing is shorter. The best combination is as followed:the first time of STZ injection (85mg/kg) is at the third week and twice totally.2. Provide a cheap and reliable platform for the evaluation and selection of drugs have used or will use in typeⅡdiabetes. |
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