| Objective: Currently, the global incidence of diabetes increased year by year.Diabetes has become one of the major chronic diseases of the 21st century, which threaten to human health. Diabetic complications have become a very important public health problem. Diabetic nephropathy (DN) is serious microvascular complications of diabetes. The occurrence and development of DN are the results of multiple factors in diabetes. The concept that over production of peroxides include reactive oxygen species (ROS) and reactive nitrogen clusters (RNS) induced by oxidative stress play an important role in the diabetes pathogenesis and its complications were widely accepted. High glucose may be oxidized by its own sugar, protein non-enzymatic glycation, increased activity of polyol pathway, protein kinase C (protein kinase C, PKC) activation in a variety of ways to cause reactive oxygen accumulation. Because excessive production of reactive oxygen species that induce expression increasing of endothelial nitric oxide synthase (iNOS), and a large number of induced nitric oxide (NO) were produced Superoxide anion ( ) were released from activation of inflammatory . NO rapidly combined with to generate peroxynitrite anion (ONOO-) which has higher oxidation ability. ONOO-can cause protein tyrosine residues or free tyrosine to nitrify and form product 3 - nitro-casein acid (3-NT) finaly. The nitro-tyrosine (nitrotyrosine, NT) is considered to be a specific marker of protein nitration. 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 the activation of nuclear factor-κB (NF-κB), and effectively inhibit the oxidative damage induced by the over activity of NF-κB which may reduce the expression of a variety of inflammatory mediators, thereby impair vascular endothelial cell function.A number of studies have shown that PDTC can reduce blood glucose levels in diabetic rats. In the current study, we observed iNOS expression, and NT production in renal cortical of type 2 diabetes rat model induced by long-term high-fat feeding accompanied with intraperitoneal injection of streptozotocin (STZ), and also studied the effects of PDTC on reduce blood glucose level and expression of iNOS, generation of NT in renal cortica, to explore the new pathway to treat diabetic nephropathy.Methods: Male, 8-week-old Wistar rats from Hebei Medical University Animal Centre weighing about 200g 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 maketype 2 diabetic model. Then the diabetic rats were further divided randomly into 2 groups: T2DM group (DM group) was fed the high-fat diet continuously a week with no PDTC treatment, and PDTC-treated group (PDTC group) was fed a high-fat diet, meanwhile PDTC was administered via intraperitoneal injection at 50mg/kg once daily. It remained 12 rats in NC group, 12 rats in DM group, and 12 rats in PDTC group at the end of experiment.All rats were weighed and taken blood at the beginning, before STZ injection and the 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 experiment day, and the blood serum was separated and stored at -20℃. At the end of experiment, the renal cortex was taken and fixed for histological analysis.1 Assays of blood glucose and insulin We measured fasting glucose by glucose oxidase method, and insulin by ELISA on serum samples.2 Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) OGTT assays: The blood glucose levels were examined at 0, 30min, 60min, 90min, and 120min after intragastric administration glucose (2g/kg).ITT assays: The blood glucose levels were assayed at 0, 15min, 30min, 60min, and 90min after intraperitoneal injection at 1IU/kg insulin.3 Urinary albumin / creatinine determination Microalbuminuria was examined by rapid immune turbidimetry, and urinary creatinine was measured with automatic biochemical analyzer.Then in urine albumin / creatinine ratio was estimated.4 Assay of blood fat Triglyceride (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.5 Assays of SOD, GSH-PX, MDA activity Tissue homogenate was prepared by centrifuging in 3000r/min for 10-15min at 4°C, supernatant was collected, and SOD, GSH-PX and MDA were assayed according the guidline of the kits.6 The Morphological detection of renal cortical 6.1 The HE-staining of renal tissuses The paraffin sections of each group were made by routine method and proceed with HE-staining. Under the light microscope, we observed the pathological morphological changes. 6.2 Observation of renal tissues through transmission electron microscopy Rat renal cortex biopsy was fixed with 4 % glutaraldehyde, ultrastructural changes of glomerular were observed though transmission electron microscope. 6.3 Immunohistochemical staining of renal cortical Renal cortex of rats in each group were selected for paraffin sections, respectively, INOS and NT content were estimated in reanal tissues throgh fluorescence microscope.6.4 Immunofluorescence staining of renal tissuse Renal tissuse was prepared by conventional frozen section, and then immunofluorescence staining was perfomed. INOS and NT content were estimated in reanal tissues throgh fluorescence microscope.7 Statistical evaluationsAll 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. 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 The parameters of each group before STZ injection The 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). 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).Levels of plasma lipids: After 8 weeks, the triglyceride, highdensity lipoprotein-cholesterol, total cholesterol, lower density lipoprotein-cholesterol in HFD group were all obviously higher than those in NC group (P<0.05) OGTT: Before injecting of STZ, the blood glucose levels at each time point in HFD group were higher than those in NC group, and the blood insulin levels at each time point in HFD group were higher than those in NC group. ITT: The blood glucose levels at each time point in HFD group were higher than those in NC group, and at the time of 15min, 60min and 90min has statistically significant different( P<0.05 ).2 The parameters of each group after STZ injection At 72h after STZ injection, the fasting blood glucose level in 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 (11.55±2.89mmol/L) in PDTC group was lower than that in DM group (26.55±2.90mmol/L) (P<0.01), but it was still higher than that in NC group (P<0.05).3 The parameters of each group after treatment with PDTC After treatment with PDTC, OGTT and ITT were were done. The blood glucose levels at each time point in PDTC group were lower than those in DM group. The blood glucose levels at each time point in PDTC group were lower than those in DM group after injecting insulin. After treatment with PDTC, the renal tissue 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 renal tissue GSH-PX activity of DM group was significantly lower than that of NC group (P<0.05), and the GSH-PX activity of PDTC group was higher than that of DM group (P<0.01).After treatment with PDTC, the renal tissue 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).4 The observation with light microscropeUnder light microscrope, HE-staining showed that rats Bowman capsule (bowmans's capsule) and capillary loop gap exists, capillary loop open, tubular structure is clear in NC group. In DM group, mesangial matrix increased, balloon adhesions, visible portion of the tubular epithelial cell degeneration, atrophy and necrosis were observed. Compared with DM group, PDTC group showed that tubular and glomerular damage were mitigated.5 Observation of rat glomerulus under transmission electron microscope NC group:It was shown that no basement membrane thickening, no fusion of foot processes, and podocyte neat were observed in the NC group kidney tissues.DM group:It was shown glomerular basement membrane thickened, uneven thickness, broken layers, fusion of podocyte processes.PDTCgroup: The morphological changes were significantly reduced, it was shown mild glomerular basement membrane thickening, mild foot process fusion.6 The detection with immunohistochemistry and immunofluorescenceAfter treatment with PDTC, The iNOS and NT expression in reanal tissues of diabetic rats were observed with immunohistochemistry and immunofluorescence. The iNOS and NT positive signals are mainaly presenced in glomerular. The signal intensity in DM group was significantly higher than that in NC group, and the iNOS and NT expressions in PDTC group was significantly lower than those of DM group.Conclusions:1 The type 2 diabetic rat model could successfully induced by long-term high-fat diet feding accompanied with intraperitoneal injection of streptozotocin (STZ).2 High Blood glucose can induce oxidative stress, and lead imbalance between oxidation and antioxidant system. The expressions of iNOS and NT in reanal tissues were significantly increased separately, as a result causing glomerular mesangial cells and epithelial cell lesions. It suggested that oxidative stress plays an important role in diabetic nephropathy.3 PDTC could reduce blood glucose levels, attenuate production of iNOS and NT in kidney tissuse, and delay occurring of microangiopathy in diabetic rats. |
PDF Full Text Request