| Objective: Diabetic cardiomyopathy (DC) is a distinct primary disease process, independent of coronary artery disease, which leads to heart failure in diabetic patients. Diabetic cardiomyopathy is often to be ignored because it has no specific symptoms at earlier period. Therefore, it is important to reduce the mortality of diabetic patients by clarifing the pathogenesis of DC, and early intervention to prevent its development. Currently, the pathogenesis of DC involving metabolic disorders, abnormal structure, pathophysiologic changes, but is still unclear. Lately, some scholars introduce"theory of oxidative stress". High glucose can increase the production of reactive oxygen species, in myocardial mitochondria. The antioxidant defense system can not completely eliminate free radicals, or loss of its defensive system due to excessive production of reactive oxygen species, leading to homeostasis imbalance.Excess amount of nitric oxide (NO) producing through the inducible nitric oxide synthase (iNOS) over expression has cytotoxic effect. NO reacts with superoxide anion (?) from inflammatory cells to generate peroxynitrite (ONOO-), which is a more potent oxidant. ONOO- makes the nitration of protein tyrosine residue, and finally produces nitrotyrosine (NT). Protein nitration, leading to the loss of enzyme activity and the cell necrosis and apoptosis, is involved in the occurrence and progression of diabetic complications.Pyrrolidine dithiocarbamate (PDTC) is a dithiocarbamates, which is a thiol-containing compound that can chelate various metal ions and has anti-oxidant properties. It can specifically suppress the activity of nuclear factor-κB (NF-κB), and efficiency inhibits the expression of a variety of inflammatory mediators caused by the activation of NF-κB in the progress of oxidative damage. A number of studies have shown that PDTC can reduce the blood glucose level in diabetic rats. The aim of the present study was to observe the effect of oxidative stress on the occurrence of DC, and the protective effects of PDTC on cardiomyopathy in type 2 diabetic rats induced by long-term high-fat feeding with intraperitoneal injection of low dose of streptozotocin (STZ). It will provide experimental basis for the development of new drugs preventing diabetes and its complications.Methods: Male Wistar rats (n=37), 8 weeks age, and weighing about 180-210g, were divided randomly into 2 groups: normal control diet (NC) group (n=12) and high-fat diet (HFD) group (n=25). After 8 weeks being fed, when the high-fat diet animals appeared insulin resistance, evaluating by oral glucose tolerance test. STZ was administered via intraperitoneal injection at 27mg/kg to establish diabetes model. NC group was administered the same volume citric acid buffer solution, and each group rats were feds with original forage. After 72 hours diabetes mellitus rats were confirmed by the random glucose over 16.7mmol/L. Then the diabetes model group was divided randomly into 2 groups: type 2 diabetes mellitus (T2DM) group and PDTC-treated group. DM group was fed the high-fat diet continuously for a week, and PDTC-treated group was fed a high-fat diet meanwhile PDTC was administered via intraperitoneal injection at 50mg/kg once daily continuously for a week. NC and DM group were given the same volume of normal saline.All the rats were monitored weight and blood glucose each week. Before injecting STZ, each group rats were done the oral glucose tolerance test (OGTT). The rats were fasted at 20:00 PM in the day before experiment, blood was taken from the angular vein at 8:00 AM of experiment day, and the blood serum was separated and stored at -20℃for detecting fasting plasma insulin, blood glucose and fat. After treated with PDTC one week all the rats were sacrificed, the myocardium tissue was taken and fixed for measureing activities of SOD, GSH-Px and content of MDA, apoptosis rate of myocardial cells, protein expression levels of iNOS and NF-κB, and morphology of myocardial cells. 1 Measurement of blood glucose and insulinFasting blood glucose was measured by glucose oxidase method, and insulin was measured by ELISA on plasma samples. Homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated to evaluate the resistance and sensitivity of insulin.2 Oral glucose tolerance test (OGTT)OGTT: The blood glucose and insulin level of 0, 30min, 60min, 90min, 120min were evaluated via intragastric administration at 2g/kg glucose.3 Measurement of blood fatTriglyceride (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), lower density lipoprotein-cholesterol (LDL-C) were detected by automatic biochemistry analyzer. Free fatty acid was measured by Cu2+ chromatometry.4 Assay of activities of SOD, GSH-Px and content of MDA in myocardiumMyocardium tissue (200mg) was homogenated with normal saline (1:9), using refrigerated centrifuge 3000r/min to centrifuge 10-15min, remaining supernatant, and to assay activities of SOD, GSH-Px and content of MDA according to their kits.5 The morphological examination of myocardial cellsThe paraffin section was made by routine method, and it was stained with HE to observed general appearance of cadiocyte, and with the method of immunohistochemistry to examine the expression of iNOS and NT producing in myocardium. The electron microscope section of each group was made after 4% glutaraldehyde fixed 24 hours, under transmission electron microscope, we observed cadiocyte ultrastructural changes.6 Assay of apoptosis rate of myocardial cellMyocardium was fixed by 70% ethanol, preparation of single cell suspension, staine 30min at 4℃with PI, then determining the apoptosis rate of myocardial cell by flow cytometry (FCM).7 Western-blotProtein was extracted with RIPA and Cytop1asmic and Nuc1ear Protein Extraction Kit, detecting the protein expression levels of iNOS and NF-κB by Western blot.8 Statistical analysesAll data were presented as means±SD. Statistical analysis was performed using SPSS 13.0. The difference between two groups was compared by the Student's t-test, and multi groups were compared by one way-ANOVA. P-value less than 0.05 was considered statistically significant.Results:1 Comparison of each group before STZ injectionThe rats were divided randomly into NC group and HFD group, there was no significant difference in weight. After 8 weeks, the weight of rats in HFD group(361.92±19.22g) was significantly higher than that in NC group(313.17±19.95g) (P<0.01).After 8 weeks, there was no significant difference in fasting blood glucose between HFD and NC group; The insulin level of HFD group(18.01±2.63μIU/ml) was higher than that of NC group(10.67±0.83μIU/ml) (P<0.05); The HOMA-IR of HFD group(1.22±0.17) was higher than that of NC group(0.68±0.09) (P<0.05). The ISI of HFD group(-4.43±0.17) was lower than that of NC group(-3.79±0.09) (P<0.05).After 8 weeks, the plasma lipids level of HFD group was higher than that of NC group (P<0.05).After 8 weeks, The blood glucose level of HFD group in each time point from OGTT was higher than that of NC group, which there was significant difference on 90min and 120min (P<0.05); The insulin level of HFD group in each time point was higher than that of NC group (P<0.05).2 Comparison of each group after STZ injectionAt 72h after STZ injection, the fasting blood glucose level of HFD group(26.16±3.38mmol/L) was significantly higher than that of NC group (4.41±0.56mmol/L) (P<0.01). 24 rats of HFD group were identified type 2 diabetes as random blood glucose>16.7mmol/L. After treatment with PDTC, the fasting blood glucose of PDTC group(11.55±2.89mmol/L) was lower than that of DM group(26.55±2.90mmol/L) (P<0.01).After treatment with PDTC, the activities of SOD and GSH-Px in DM group were lower than those in NC group (P<0.05), and the activities of SOD and GSH-Px in PDTC group were higher than those in DM group (P<0.05); The content of MDA in DM group was higher than that in NC group (P<0.05), and the content of MDA in PDTC group was lower than that in DM group (P<0.05).Myocardic tissues stained with HE were observed. In NC group: myocardic structure was clear, the numbers of myocardial cells ranks tight, and cytoplasm evenly. In DM group: Disordered arrangement of myocardial fibers, myocardial cell degeneration and necrosis, fat cells and inflammatory cells infiltration between myocardial cells were found. In PDTC group: Myocard structure was obviously improved after treatment with PDTC.Observing by immunohistochemistry, the expression of iNOS and production of NT in DM group were higher than those in NC group, and the expression of iNOS and production of NT in PDTC group were lower than those in DM group.Cadiocyte ultrastructure with TEM was observed. In NC group: myocardic sarcomere tidy, mitochondria well-structured. In DM group: Sarcomere dislocation, cristae and membrane of mitochondria, Medullary of mitochondria, endoplasmic reticulum, accumulation of glycogen and lipid were found. In PDTC group: Myocard structure was obviously improved after treatment with PDTC.The apoptosis rates of myocardial cells were detected by FCM. The apoptosis rate of myocardial cells in DM group(25.88±1.63%) was higher than that in NC group(18.75±3.47%) (P<0.01), while the apoptosis rate of myocardial cells in PDTC group(20.99±4.01%) was lower than that in DM group (P<0.01).Protein expression levels were assaied by western-blot. The protein expression level of iNOS in DM group was higher than that in NC group, and the protein expression level of iNOS in PDTC group was lower than that in DM group. The protein expression level of NF-κB has no significant difference in total protein. In nucleus, the protein expression level of NF-κB in DM group was higher than that in NC group, and the protein expression level of NF-κB in PDTC group was lower than that in DM group.Conclusions:1 The type 2 diabetic rat model could be successfully established by long-term high-fat diet fedding with a small dose of intraperitoneal injection of STZ.2 Hyperglycemia induced oxidative stress, results in imbalance of oxidative and antioxidant systems, increasing the expression of iNOS and production of NT through activation NF-κB signaling pathway, and leading to apoptosis of myocytes. All of those indicate that oxidative stress plays an important role in diabetic cardiomyopathy.3 PDTC not only can reduce blood glucose levels, but also has a good protection on diabetic cardiomyopathy in diabetic rats. So early using of PDTC can prevent diabetic cardiomyopathy.
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