| Objective: Diabetes has become one of the major chronic diseases of the 21st century, which threats to human health. Diabetic macroangiopathy is main reason for the diabetic's mutilation and early death, more than 70% of diabetic patient died of diabetic macroangiopathy. A lot of evidences indicated that diabetic macroangiopathy is relation to excess peroxynitrite recently. Hyperglycemia produced excess amount of nitric oxide (NO) through the inducible nitric oxide synthase (iNOS). Excess amount of NO has cytotoxic effect. NO reacts with superoxide anion ( ) to generate peroxynitrite (ONOO-), which is a more potent oxidant. ONOO- makes the tyrosine of protein or free tyrosine nitration, finally producing nitrotyrosine(NT), which is an indicator of peroxynitrite production. Protein nitration can induce the loss of enzyme activity, cell necrosis and apoptosis. Pyrrolidine dithiocarbamate (PDTC) is a dithiocarbamates, which is a thiol-containing compound that can chelate various metal ions and have anti-oxidant properties. It can specificly suppress nuclear factor-κB (NF-κB), and effectively inhibit the oxidative damage induced by the expression of NF-κB, reducing the expression of a variety of inflammatory mediators, thereby improving vascular endothelial cell function.A number of studies have shown that PDTC can reduce blood glucose levels in diabetic rats. In the current study, The aim of the present study was to examine the expression of iNOS and NT in aortic endothelium in type 2 diabetic rats induced by long-term high-fat feeding accompanied with intraperitoneal injection of streptozotocin (STZ), and the effects of PDTC on reducing blood glucose levels and production of iNOS and NT.Methods: Male 8-week-old Wistar rats from Hebei Medical University Animal Centre weighing 180-210g were used. A total of 37 healthy male Wistar rats were divided randomly into 2 groups: normal control group (NC group) received a regular diet and high-fat diet group (HFD group) received high-fat diet was fed for 8 weeks. When the high-fat diet animals appeared insulin resistance, STZ was administered via intraperitoneal injection at 27mg/kg to make model. Then the modeling group were further divided randomly into 2 groups: T2DM group (DM group) was fed the high-fat diet continuously a week, and PDTC-treated group (PDTC group) was fed a high-fat diet meanwhile PDTC was administered via intraperitoneal injection at 50mg/kg once daily. There remain 12 rats in NC group, 12 rats in DM group, 12 rats in PDTC group at the end of experiment.All the rats were weighed and taken blood at the beginning, before STZ injection and the end of the experiment. The rats were fasted at 20:00PM in the day before experiment, blood was taken from the angular vein at 8:00AM of experiment day, and the blood serum was separated and stored at -80℃for later analysis. At the end of experiment, the vascular tissues were taken and fixed for histological analysis.1. Measurement of blood glucose and insulinPlasma fasting glucose were measured by glucose oxidase method, and insulin were measured by the use of a commercially available ELISA kit.2. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)OGTT assays the blood glucose at 0, 30min, 60min, 90min, 120min after intragastric administration glucose at 2g/kg. ITT assays the blood glucose at 0, 15min, 30min, 60min, 90min after intraperitoneal injection at 1IU/kg insulin.3. Measurements of blood lipidsTriglyceride (TG), highdensity lipoprotein-cholesterole (HDL-C), total cholesterol (TC), lowerdensity lipoprotein-cholesterole (LDL-C) were detected. Free fatty acid (FFA) was measured by Cu2+ chromatometry.4. Analysis of the activity of SOD, GSH-PX and MDA in aortasPrepare tissue homogenate, using refrigerated centrifuge 3000r/min to centrifuge 10-15min, remaining supernatant, and assay SOD, GSH-PX and MDA according to their kits. 5. Histological measurementsThe paraffin section of each group was made by routine method and proceed with HE staining. Under the light microscope, we observed the pathological morphological changes.6. Histochemistry detection of iNOS and NTPrepared the paraffin section, and proceed with immunohistochemistry.7. Immunofluorescence detection of iNOS and NTThe paraffin section was made by routine method and proceed with immunofluorescence. Images were taken by immunomicroscope.8. Statistical analysesAll data were presented as means±SD. Statistical analysis was performed using SPSS 13.0. A 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 statistically significant difference in weight and fasting blood glucose at the beginning of the experiment.After 8 week, 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). There was still no statistically significant difference in fasting blood glucose. After 8 week, the insulin level of HFD group (18.01±2.63μIU/ml) was obviously higher than that of NC group (10.67±0.83μIU/ml)(P<0.01). 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 week, the serum lipid level of HFD group was higher than that in NC group (P<0.05).After 8 week, the result of OGTT was that the blood glucose level of HFD group in every time was higher than that of NC group. The result of ITT was that the blood glucose level of HFD group in every time was higher than that of NC group.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 in NC group (4.41±0.56mmol/L)(P<0.01). 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), but it was still higher than that of NC group (P<0.05).After treatment with PDTC, the result of OGTT was that the blood glucose level of PDTC group was lower than that of DM group in every time. After treatment with PDTC, the result of ITT was that the blood glucose level of PDTC group was lower than that of DM group in every time.After treatment with PDTC, the SOD activity of DM group was significantly lower than that of NC group (P<0.01), and the SOD activity of PDTC group was significantly higher than that of DM group (P<0.01).After treatment with PDTC, the GSH-PX activity of DM group was significantly lower than that of NC group (P<0.01), and the GSH-PX activity of PDTC group was higher than that of DM group (P<0.01).After treatment with PDTC, the MDA activity of DM group was significantly higher than that of NC group (P<0.01), and the MDA activity of PDTC group was significantly lower than that of DM group (P<0.01).Under light microscrope, HE-staining showed that the vessel wall smooth and glossy, the endothelium integrity and smooth muscle arranged in neat rows, the cell outline clear in NC group. In DM group we observed that arterial intima interrupted, endothelium intermittent, smooth muscle disarray, interstitial edema. Compared with DM group, PDTC group showed that endothelia surface and the smooth, endothelial defects and interstitial edema significantly reduced.After treatment with PDTC, the result of immunohistochemistry and immunofluorescence is that the iNOS and NT expression of the rats'aortae endothelium fluorescence intensity in DM group was significantly higher than that in NC group, and the iNOS and NT expression of PDTC group was significantly lower than that of DM group.Conclusions:1 The type 2 diabetic rat model could successfully induced by long-term high-fat diet fedding, accompanied with intraperitoneal injection of streptozotocin (STZ).2 Impairment of blood vessels defense from oxidative damage in type 2 diabetic rats, imbalance of redox, meanwhile over expression of iNOS and NT in vascular endothelial cell demonstrate that oxidative stress play an important role in diabetic macroangiopathy.3 PDTC could reduce blood glucose levels, attenuate production of iNOS and ONOO- in aortic endothelium and delay macroangiopathy in diabetic rats. So early application of PDTC can prevent diabetic macroangiopathy from "metabolic memory". |
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