| Objective: The morbidity rate of macroangiopathy in diabetic complication is 3 to 5 times of non-diabetic. It is the major complication of diabetic and main reason for the diabetic's mutilation and early death. The abnomal metabolism of the diabetics cause the dysfunction of arteries. The main modules such as long-term hyperglycemia, hypertension, dyslipidemia, insulin resistence, lead to diabetic complicating with macroangiopathy. But atherosclerosis(AS) is the chief process of macroangiopathy. The closely relationship between diabetic and atherosclerosis show that the diabetics occurs AS earlier and more seriously, and they have bad prognosis. The recent study shows that matix metalloproteinases(MMPs) is related to the development of diabetic complicating with AS. Peroxisome Proliferator-activated receptor-γ(PPARγ) , a member of the nuclear hormone receptor superfamily, play a critical role in lipid and lipoprotein metabolism and glucose metabolism. Thiazolidinediones (TZDs), a kind of high affinity ligand of PPARγ, through activating PPARγ, effectively improving insulin resistence, cutting down the blood glucose, retrieving the lipid metabolic disturbance, is generally utilized in the treatment of T2DM. The research recently show that PPARγhas involved in arteriovascular inflammatory reaction and the development of AS. The activation of PPARγcan protect the vessel wall directly. As a PPARγagonist may be significance to the treatment of chronic inflammation disease,especially to diabetic macroangiopathy because of its inhibitory action to blood vessel inflammation. Pioglitazone is a kind of new drug belonging toTZDs. Pioglitazone has the founctions that not only lowering the hyperglycemia, improving lipid metabolic disturbance, but also depressing the hypertension, reducing albuminuria, and anti-atherosclerosis. In the diabetic macroangiopathy, the effect of Pioglitazone on expression of MMP-1 and TIMP-1 on the vessel wall is not report yet. In the present study,we investigate the observation that the expression of MMP-1,TIMP-1 in rat of diabetic macroangiopathy model induced by long-term high-fat feeding accompanied with intraperitoneal injection of streptozotocin (STZ), and also research the effect of Pioglitazone on expression of MMP-1 and TIMP-1,changes of the collogen content in the vascular, and alteration of the functions of endothelium, explored the mechanisms of diabetic macroangiopathy treatment by PPARγagonist.Methods:Male Sprague–Dawley rats (Hebei Medical University) weighing about 216±15g (about 8 weeks old), were used. The rats were housed under 12 h light/12 h dark conditions with adlibitum access to water and food. After breeded adaptively for 1 weeks,a total of 45 healthy male SD rats were divided randomly into 2 groups: normal control group (Group NC, n=15) received a regular diet and experimental model group (n =30) was fed for 1 months. When the high-fat diet animals appeared the insulin resistance evaluated by Li Guangwei HOMA index formula , STZ was administered via intraperitoneal injection at 30mg/kg to make model. Then the modeling rats were further divided randomly into 2 groups: non-intervention of T2DM group (Group DM, n=15) was fed the high-fat diet continuously for 2 months, and Pioglitazone -treated group (Group PIO, n=15) was fed a high-fat diet meanwhile Pioglitazone was administered by oral gavage (11mg/kg) once daily. The experiment lasted 14 weeks. Except for accidental death and abortive sample, there remain 15 rats in Group NC,12 rats in Group DM and 13 rats in Group PIO at the end of experiment.All the rats were weighed and their blood was taken at the beginning, before injecting STZ and end of the experiment. The rats were fasted at 20:00 in the day before experiment, blood was taken from the angular vein at 8:00 of experimental day, and the blood serum was separated and stored at -20℃for all the indexes'detection (the mensuration of the indexes were performed with the same batch of kits). At the end of experiment, the vascular tissues were taken and fixed for histological analysis by neutral paraform. 1 Assays blood glucose and insulinWe measured fasting glucose by glucose oxidase method, and insulin by radioimmunoassay on serum samples. The level of insulin resistance was evaluated according to Homeoestasis Model Assessment (HOMA-IR) and Insulin Sensitivity Index (ISI).2 Assays of blood fatTriglyceride(TG), total cholesterol(TC) were detected. FFA was measured by Cu2+chromatometry.3 Assays of vascular parametersEndothelin(ET) was measured by radioimmunoassay on serum samples. NO measured by nitrate reductase method.4 Cell morphology examination of vessel wallThe tissue section was made by routine method for light microscope examination, and proceed with HE staining,masson staining, MMP-1 and TIMP-1 immunohisto-chemistry staining. Observed vessel wall microstructure with light microscopes. Measured vessel wall collagen content by picric acid - brilliant green staining. Detected the protein expression level and activity of MMP-1 and TIMP-1 in vessel wall by immunohistochemical staining. hese graphic results were analyzed with computer image-analysis system and the integral optical density (IOD) counts of MMP-1,TIMP-1 and collagen content were calculated.5 Data analysis and statistical evaluationAll data were treated with SPSS 13.0. Measurement data were expressed as mean values±standard deviation. The difference between means was compared by the Student's t-test, and analysis of variance for one factors (ANOVA) was used to compare continuous variables among groups. The degree of significance was judged by P values when necessary. P<0.05 means the difference is significant , P<0.01 means the difference is highly significant.Results:1 Body weight: There was no statistically significant difference in weight among each group of the rats at the beginning of the experiment. At the end of the experiment, the weight of rats in diabetic rats and was significantly higher than that in normal control rats (P<0.01), and the weight of rats treated with Pioglitazone was lower than that diabetic rats(P<0.05).2 Blood glucose and insulin: There was no significant difference in fasting glucose among the rats of each group at the beginning of the experiment. At 2 weeks after the injection of STZ, the blood glucose level of rats in Group DM and the rats in Group PIO significantly raised up from normal baseline (P<0.01). The blood glucose level of rats in Group PIO was lower than that in Group DM (P<0.05), but it was still hyperglycemia. The insulin level of rats in Group DM was markedly higher than that in Group NC (P<0.01), The insulin level of rats treated with Pioglitazone lower than that in Group DM and higher than that in Group NC(P<0.05).3 Blood lipid :The plasma levels of TG, TC, FFA in rats with T2DM were obviously higher than those in normal control rats (P<0.01). After treated with Pioglitazone,their plasma levels decreased to some extent, but they were still higher than those in normal control rats.4 vascular parameterThe plasma levels of ET in rats with T2DM were obviously higher than those in normal control rats(P<0.01). The plasma levels of ET in rats treated with Pioglitazone was lower than that in rats with T2DM(P<0.05), but still higher than that in normal control rats(P<0.05). And NO in rats with T2DM were obviously lower than those in normal control rats(P<0.01). The plasma levels of NO in rats treated with Pioglitazone was higher than that in rats with T2DM(P<0.05), but still lower than that in normal control rats(P<0.05).5 The pathological morphological change of the vascular tissuesHE-staining method: under light microscope, we observed that, compared with the Group NC,the vascular tissue of the diabetic rats show significantly thicken in local tunica intima,but can't find the typical pathological change of AS, such as a great of foam cells.Masson-staining method: Under light microscope, the intima shows a little green in normal control rats, while the green area in Group DM and in Group PIO was bigger than that in Group NC (P<0.05),the green area in Group PIO was smaller than that in Group DM (P<0.05).6 Immunohistochemistry The MMP-1 and TIMP-1 expression of the rats treated with Pioglitazone was significantly higher than that of normal control rats (P<0.05), and lower than that of diabetic rats (P<0.05).Conclusions:1 Male SD rat was fed with a diet enriched with sucrose (20%),cholesterol(2.5%) and lard(15%) to induce insulin resistance, and after two month on the diet streptozotion(STZ,30mg/kg) was injected in abdominal cavity to induce hyperglycemia. The rat model was succeed in preparing for T2DM , with hyperglycemia, insulin resistance and dyslipidemia. It also shows that there exists early stages of atherosclerosis in the model of T2DM rats.2 The disturbance of MMP-1 and TIMP-1 activation and expression in diabetic rats constitutes the development of atherosclerosis,as one of the critical factors.3 Pioglitazone plays an important role in improving glucolipid metabolism, increasing the sensitivity of insulin and reducing the insulin resistance,by activating PPARγ. Moreover, it can regulate the expression of MMP-1 and TIMP-1 in vascular tissues, increase the activity of NO, reduce the activity of ET. Thus it exerts the action against inflammatory reaction and oxidative stress , improves the blood vessel endothelial function, protects vascular tissues in T2DM, and slows the process of diabetic atherosclerosis.
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