The Expression Of MMP-1,TIMP-1 And Collagen In Myocardium Of T2DM Rats And The Protective Effects Of Pioglitazone

Objective: Diabetic cardiomyopathy (DC) is one kind of unique cardiovascular complications of diabetes mellitus and it has a close relation with the high rate of heart failure and death in diabetes mellitus (DM) patients. Diabetic cardiomyopathy is a special chronic complication of DM, and its major pathological hallmarks of DC were myocardial hypertrophy, myocardial necrosis, myocardium mesenchyme remodeling, myocardial fibrosis and so on. Recent years, a lot of research revealed that myocardium mesenchyme remodeling play an important role in DC. Extracellular matrix (ECM) is the principal part of myocardium mesenchyme, and keeps the balance in metabolic of continuous synthesis, secretion and degradation. Matrix metalloproteinases (MMPs) exist in myocardial tissue; they are the main enzymes to degrade all the components of ECM. The change of MMPs and their tissue inhabitor -(TIMPs)will make ECM lose its balance. The changes of MMPs and TIMPs protein expression levels and accounts of collagen may play the key role during the occurrence and development of cardiomyopathy in DM.Pioglitazone that belongs to thiazolidinediones (TZDs) is a kind of new drug treating T2DM through activating PPARγ. More and more studies pay attention to thiazolidinediones because their potencial protection to many body organs.In this study type 2 DM (T2DM) rat model was copied by giving Sprague-Dawlay (SD) rats long-term high-fat diet and a low dose intraperitoneal injection of streptozotocin (STZ). Then we detected the protein expression level and activity of MMP-1,TIMP-1 and the content of collagen (CL) in experimental animals myocardial tissue to know the connection with the change of blood glucose and blood lipid in order to discuss the mechanism of DC.Furthermore, Pioglitazone was given to experimental rats to observe the function to MMP-1,TIMP-1 for explaining the mechanism of its protective effects on myocardium .Methods:1. Induction of type 2 diabetic cardiomyopathy in ratsWe chosen 45 healthy male SD rats as experiment objects .After breeded adaptively for 1 week, they were divided randomly into 2 groups: control group (Group A,n=15) fed regular diet and experiment group (n =30) fed high-sugar and high-fat diet (including 2.5% cholesterin, 15% cooked lard, 20% cane sugar and general diet together) for 4 weeks. When the high-fat diet animals appeared the insulin resistance according to Li Guangwei HOMA index formula, STZ was administered via intraperitoneal injection at a dose of 30mg/kg once. Two weeks later, the rats whose fasting blood glucose (FBG) was greater than 7.8mmol/L and insulin sensitivity decreased were considered as T2DM model rats. The model rats were divided randomly into two group: DM model rats (group B) fed the high-sugar and high-fat diet continuously for 8 weeks, and DM model rats treated with Pioglitazone (Group C) which fed a high-sugar and high-fat diet meanwhile pioglitazone was administered by oral gavage (11mg/kg) once daily for 8 weeks. Except for accidental death and abortive sample, there remain 15 rats in A group, 12 rats in B group, 13 rats in C group at the end of the experiment. All the rats were weighed and their blood was taken at the beginning 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.At the end of experiment, the heart tissues were taken and fixed for histological analysis by neutral paraform.2 Assays of 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).3 Assays of blood lipidAt the end of this experiment, Triglyceride (TG), total cholesterol (TC) was measured by chromometry. FFA was measured by Cu2+chromatometry.4 Cell morphology examination of myocardiumThe tissue section was made by routine method for light microscope examination, and proceeds with MASSON specific staining to calculate the content of CL. Detecting the protein expression level and activity of MMP-1 and TIMP-1 in myocardium by immunohistochemistry.5 Data analysis and statistical evaluationAll data were treated with SPSS11.0. 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. In pykno-ranked data, the difference between two groups was compared by the Wilcoxon's rank test, and Kruskal-Wallis H's test was used to compare the difference 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 group B and group C was significantly higher than that in group A (P<0.05), and the weight of group C was lower than that of group B (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. After the injection of STZ, the blood glucose level of model rats and the rats treated with pioglitazong significantly raised up from normal baseline (P<0.01). The blood glucose level of rats treated with pioglitazong was lower than that of model rats (P<0.01), but it was still hyperglycemia. The insulin level of model rats was markedly higher than that of rats treated withpioglitazong and control rats (P<0.01), The insulin level of control rats lower than that of rats treated with pioglitazong (P<0.05).3 Blood lipid: The plasma levels of TG, TC and FFA in rats with T2DM were obviously higher than those in control rats (P<0.01). After treated with pioglitazong, their plasma levels decreased to some extent, but they were still higher than those in control rats(P<0.01).4 Immunohistochemistry: The MMP-1 expression of the rat myocardium in group C was significantly higher than that in group A (P<0.01), and lower than group B (P<0.01). The TIMP-1 expression of the rat myocardium in group C was significantly higher than that in group A (P<0.01), and lower than that in group B (P<0.01). The ratio of TIMP-1/ MMP-1 in group C was significantly higher than that in group A (P<0.05), and lower than group B (P<0.05).5 The pathological morphological change of myocardiumHE staining showed that cardiac tube of group A were in order, cross striation was clear, karyon and ECM was normal. While in T2DM model rats, cardic muscle degeneration and ECM accumulation could be observed. Foregoing changes in-group B rats were more severe than thosein group C rats.MASSON-staining method: Under light microscope, most of cardiac muscle fibers were stained with red, only few collagens showed green in control rats. There was more collagen showed green in group B than groupA, less than group C.Conclusions:1 The type 2 diabetic rat model that was induced by high-sugar, high-fat diet and a low dose of STZ had some characteristics similar to human T2DM. It would be available to study T2DM and its complications.2 The protein expression level and activity of MMP-1,TIMP-1 in T2DM rats myocardium was increased. These changes lead collagen hyperplasia and ECM remodeling. It indicated that the balance of TIMP-1/MMP-1 was an important factor to the occurrence and development of DC.3 By activating PPARγ, pioglitazong plays an important role in improving glucolipid metabolism, increasing the sensitivity of insulin and reducing the insulin resistance. Moreover, it can lower the expression of MMP-1 and TIMP-1 in cardiac muscle, improve the balance of TIMP-1/MMP-1, thus it exerts the action against remodeling of ECM and protects cardiac muscle in T2DM, and slows the process of diabetic cardio- myopathy.