| Objective:To establish an experimental diabetic rat model with insulin resistance and dyslipidemia, and monitor continuously three batches of rat models whom was produced by the same methods, to assay the stability of the model through measuring the relevant indes.Methods:A total of 330 male SD rats in three batches, each of batches was randomly divided into experimental group and control group, control group was fed a normal diet, the experimental group fed high fat and sugar diet for 4 weeks, the experimental groups were injected with 1% streptozotocin (STZ)by two steps, the normal groups were injected with O.1mmol/L citric acid-citrate buffer which was same volume with STZ. Those rats whose random blood glucose were more than 13.8mmol/L was to become the standard models. To detecte body weight, blood glucose, serum insulin, insulin resistance index, glycated hemoglobin and blood lipids in the first batch of rat, and make clear the degree of insulin resistance and dyslipidemia of the model. Based on the first barch, used the same method and optimized relative conditions, to make the second and the third batches of diabetic rat model, by compared body weight, blood glucose, persistence and death of models with them.To clarify the stability and the advantages and disadvantages of the model method.Results:1 Insulin levels(35.39±30.63) was decreased in the experimental group:, there was statistically significant compared with the control group; insulin sensitivity was reduced, insulin sensitivity index was-2.61±0.34, there was statistically significant compared with the control group (P＜0.05); glycosylated hemoglobin (6.71±1.37%) was significantly higher compared with the control group,there was statistically significant with the control group (P<0.05).2 Blood lipids was elevated in diabetes rats,LDL was 0.75±0.32mmol/L, TC was 1.43±1.07 mmol/L, TG was 1.72±0.6mmol/L, there was statistically significant compared with the control group (P<0.05).3 The blood sugar levels was stable in three batches of rats, it was(16.61±4.20, 22.17±5.6,19.56±1.42) at 16 weeks; the success models rates were increased gradually(72%,78%,85%), the death rates were decreased (33%,28%,25%).Conclusion: 1 The high-fat diet combined with STZ can replicate the rat model with insulin resistance and progress was similar to type 2 diabetic of human. The model has high blood sugar, high blood lipids, hyperinsulinemia and insulin resistance and other features which would be benefit to neuropathy and angiopathia.It is the ideal animal mode to study angiopathia mechanism in type 2 diabetes2 The relevant indexes in three batches of diabetic rat model had consistence tendency, and the success model rates were increased gradually, that indicated the diabetic rat model was stability, can be used for related research.3, Through optimizing the relevant conditions that can increase the successed model rate and reduce mortality rate. Objective:To detect the expression levels of antioxidant enzymes (SOD, CAT, GXP), AR in the brain tissue of DM rat in different periods (8 weeks,12 weeks,16 weeks) and different blood glucose levels, combined with pathological changes, to explore the relationship among diabetic encephalopathy,antioxidant enzymes and the AR.Methods:90 diabetic rat models established in the first part were divided into six experimental group and a normal control group in accordance with the different stages (8 weeks,12 weeks,16 weeks) and different blood glucose levels (13.8< L group< 16.7 mmol/L,H group> 16.7mmol/L).Detected expression and localization of SOD, CAT, GXP, AR on the rats brain by by Western blot and immunohistochemical, and rat brain tissue pathological changes observed by electron microscopy. Analysis comprehensively the relationship among encephalopathy antioxidant enzymes and AR at different stages of diabetic pathological changes.Results:1 Eectron microscope showed brain tissue vascular endothelial cell swelling, dim nucleus, sparseness of endochylema, perithelial cell's degeneration, glassy degeneration of capillary vessel wall, crinkle collapse of vessel wall, focal hyperplasia of gliocyte and attenuation of GRL under optical microscope at the 16th week. Injury of brain blood vessel and neuron was gradually aggravated following course of disease. Pathology induced by different blood glucose concentration exhibited diversely in different groups. Pathology of H group was more severe than that of L group.2 Western blot results showed that,with the progression of disease, the expression of SOD, CAT and GPx were significant different between experimental group and control group but except for GPx 12w (p< 0.05),which showed decreased gradually,and 8w SOD was different between H and L group (p<0.05).The expression of AR was different significantly between experimental group and control group (p<0.05), which present a gradual increasing tendency with the progression of diabetes,there was different between H and L group(p< 0.05) but except for AR 12w.3 Immunohistochemistry showed the expressions of SOD,GPx and CAT in brain tissue grew down gradually with the course of disease,.and taken on the negative correlation between experimental group and control group. On the contrary,AR expressions rised gradually and taken on positive correlation. There were both expressions of GPX,CAT,SOD and AR in diabetic rats and control group rats pallium, hippocampus and blood vessels, SOD expression mainly located in kytoplasm and cellular membrane, CAT and GPX expressed in kytoplasm, and expression of AR concentrated in nucleus and nuclear membrane.Conclusion:1 There were pathological changes in brain, blood vessels, and nerve tissue of diabetic rats to different extents, and which turned to be more obvious with the developmental course of disease. Pathological changes of H group with higher blood glucose took place more early and quickly than in L group, which indicated apparent brain tissue damages correlated intimately with course of disease and blood glucose level.2. SOD,GPx and CAT expressions in brain tissue were raised up at initial stage and decreased gradually with development of course of disease, which indicated obvious ly strengthened oxidative stress in brain tissue.AR expressions i were raised up gradually with development of course of disease, it showed that fluorescence increasing of polyol pathway maybe a important machnism of diabetic encephalopathy. Which arised the difference between H and L of SOD and AR showed their glucose dependence. 3 There were evident oxidative stress and aldose reductase alteration in diabetic rat brain tissue, and they had close correlation with vasculopathy and neuropathy in brain tissue. Oxidative stress and aldose reductase promoted diabetic brain damage together. |
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