Effects Of Pyrrolidine Dithiocarbamate On Islet β Cell Apoptosis In Type 2 Diabetic Rat

Objective: The result of United Kingdom Prospective Diabetes Study (UKPDS) sugessets the function of islet in patients with newly diagnosed type 2 diabete was 50% lower than that in normal people, and as the progression of this disease,βcell function has been deteriorated. Whereas increasing of isletβcells apoptosis was one of the major reasons in the reducing ofβcell numbers, so it is very important to protect stup ofβcell to delay diabetes progression, and also it is research focus in the fild of diabetic treatment. Hyperglycemia and hyperlipemia in vivo of patients with type 2 diabetes induce oxidation and antioxidation disording and result in oxidative stress, which is a major reason to cause the impairation of isletβcells and diabetes progression. High level of blood glucose can make the cells produce excess reactive oxygen species (ROS), that induce oxidative stress and activate NF-κB signing pathway, moreover, which increase the amount of nitric oxide (NO) through the inducible transcription of nitric oxide synthase (iNOS). NO can induceβcell apoptosis through many ways. In addition, excessive NO can bind with superoxide anion (?) to generate peroxynitrite (ONOO-), which is a more potent oxidant, can impair and facilitateβcells apoptosis. ONOO- makes the tyrosine residues of protein nitration, finally producing nitrotyrosine. 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 has the function of anti-oxidant. It can specificly suppress nuclear factor-κB (NF-κB) activity, and efficiency inhibit the oxidative damage that induced by the activation of NF-κB. A number of studies have shown that PDTC can reduce the level of blood glucose in diabetic rats, but the mechanism is still unknown. In the current study, type 2 diabetic rat model were established by small dose of streptozotocin (STZ) and high-fat diet. Interventing with pyrrolidine dithiocarbamate (50mg/kg/d, IP) continuously a week, then blood glucose was evaluated, superoxide diamutase (SOD), glutathion peroxidase (GSH-Px) activity, and malonaldehyde (MDA) content in pancreatic tissues were examined, iNOS expression and NT prouduction in pancreatic tissue were detected, and the apoptosis rate of pancreatic isletβcell was measured, to explore the effects of pyrrolidine dithiocarbamate on againsting oxidative stress and nitrification stress to protect isletβcell from impairing.Methods:1 Animal care and sample collectionMale Wistar rats (n=37), 8 weeks of 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, evaluated by oral glucose tolerance test (OGTT), STZ was administered via intraperitoneal injection at 27mg/kg to make diabetes model. Control group administers the same volume citrate buffer solution, and each group rats were fed 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: T2DM group was fed with high-fat diet continuously a week, and PDTC-treated group was fed with high-fat diet meanwhile with PDTC was administered via intraperitoneal injection at 50mg/kg once daily continuously a week. Control and diabetes group were given the same volume of saline.All the rats were monitored weight and blood glucose level each week. Before or after injecting STZ, each group rats were detected with the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). 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 plasma was separated and stored for detecting fasting plasma insulin, triglyceride (TG), highdensity lipoprotein-cholesterole (HDL-C), total cholesterol (TC), lowerdensity lipoprotein-cholesterol (LDL-C), free fatty acid (FFA). After treatment with PDTC for a week, all the rats were sacrificed, and pancreatic tissue was taken for measureing the expression of iNOS and NT, and the activities of SOD and GSH-PX, the content of MDA, and the apoptosis rate of isletβcell.2 Measurement of blood glucose and insulinFasting blood glucose was measured by glucose oxidase method, and insulin was measured by a commercially available ELISA kit. Homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated to evaluate the insulin resistance and insulin sensitivity.3 Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)OGTT: The blood glucose and insulin level of 0, 30min, 60min, 90min, 120min were evaluated via intragastric administration at 2g/kg glucose, and to compute the area under the curve (AUC).ITT: The blood glucose level of 0min, 15min, 30min, 60min, 90min were evaluated via intraperitoneal injection at 1U/kg insulin.4 Blood fatTriglyceride (TG), highdensity lipoprotein-cholesterol (HDL-C), total cholesterol (TC), lowerdensity lipoprotein-cholesterol (LDL-C) were detected by automatic biochemistry analyzer. Free fatty acid was measured by Cu2+ chromatometry.5 Assay of the activities of SOD, GSH-Px and content of MDA in pancreatic tissuePancreatic tissue (200mg) was homogenated with normal saline (1:9), using refrigerated centrifuge 3000r/min to centrifuge 10-15min, remaining supernatant, and to assay the activity of SOD, GSH-Px, and content of MDA according to their kits manual.6 The morphological examination of pancreatic islet cellThe paraffin section was made by routine method, and it was stained with HE to observe general appearance of islet. The method of immunohistochemistry was used to examine the expression of inducible nitric oxide synthase (iNOS) and protein nitration (NT) prouduction in islet cell.7 Assay of the apoptosis rate of pancreatic isletβcell by flow cytometryPancreatic tissue was fixed by 70% alcohol, preparation of single cell suspension, stain 30 min at 4℃with anti-Insulin/FITC and PI, then determining the apoptosis rate of isletβcell by flow cytometry.8 Statistical methodsAll data were presented as mean±SD. Statistical analysis was performed using SPSS 13.0. The differences between two groups were compared by the Student's t-test, and multi groups were compared by one way-ANOVA. P-value less than 0.05 is considered statistically significant.Results:1 Comparison of each group before STZ injectionWe feed different forage accoding to subgroup, there is no statistically significant different in body weight between HFD and NC group at the beginning of the experiment. After 8 weeks the weight of rats in HFD group (361.92±19.22g) was significantly higher than that in control rats (313.17±19.95g) (P<0.01).After 8 weeks, before injecting of STZ, there is no significant different in fasting 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), and the ISI of HFD group (-4.43±0.17) was lower than that of NC group (-3.79±0.09) (P<0.05).OGTT: Before injecting of STZ, the blood glucose of HFD group in every time was higher than that of NC group. The area under the curve (AUC) in HFD group (27.91±2.16) was higher than that in NC group (25.65±1.44) (P<0.05), and the insulin of HFD group in every time was higher than that of NC group, all time points have significant different (P<0.05).ITT: The blood glucose of HFD group in every time was higher than that of NC group, and at the time of 15min, 60min and 90min has significant different (P<0.05). Levels of plasma lipids: After 8 weeks, the triglyceride, highdensity lipoprotein-cholesterol, total cholesterol, lowerdensity lipoprotein-cholesterol in HFD group were all obviously higher than those in NC group (P<0.05).2 Comparison of each group after STZ injectionAt 72h after injection of STZ, the fasting blood glucose of HFD group (26.16±3.38mmol/L) was significantly higher than that of NC group (4.41±0.56 mmol/L) (P<0.01). At the end of the experiment, the blood glucose of PDTC group (11.55±2.89 mmol/L) was lower than that of DM group (26.55±2.90 mmol/L) (P<0.01).After PDTC treatment, OGTT: The blood glucose level of PDTC group was lower than that of DM group in each time point, and the level of area under the curve (AUC) in PDTC group (63.06±25.27) was lower than that of DM group (101.83±16.89) (P<0.05).ITT: The blood glucose level of PDTC group was lower than that of DM group in each time point.At the end of the experiment, the activities of SOD and GSH-Px in DM group were significantly lower than those in NC group (P<0.01), and the activities of SOD and GSH-Px in PDTC group were significantly higher than those in DM group (P<0.05). At the end of the experiment, the content of MDA in DM group was significantly higher than that in NC group (P<0.01), and the content of MDA in PDTC group was significantly lower than that in DM group (P<0.01).The result of pancreatic tissue with HE-staining: In NC group: Pancreatic islet structure was clear, and pancreatic islet cells rank tight, and cell nucleus stain royal blue, clear, large, and round, and endochylema was abundant, while in DM group: Pancreatic islet structure was not clear, the numbers of pancreatic islet cells were decreased, and some islet cells becomed vacuole. In PDTC group: The numbers of pancreatic islet cells were higher than those of DM group, and pancreatic islet structure was obviously improved.Immunohistochemistry results: The expression of iNOS and production of NT in DM group were significantly higher than those in NC group (P<0.01), and the expression of iNOS and production of NT in PDTC group were significantly lower than those in DM group (P<0.01).The apoptosis rate of pancreatic isletβcell in DM group (22.44±5.15%) was significantly higher than that in NC group (10.35±1.95%) (P<0.01), while the PDTC group (12.14±4.66%) was significantly lower than that in DM group (P<0.05).Conclusions:1 The type 2 diabetic rat model could successfully was established by long-term high-fat diet fedding and intraperitoneal injecting small dose of STZ.2 Hyperglycemia and hyperlipemia induced oxidative stress, in the same time increased iNOS expression and NT production that play an important role in pancreatic isletβcell damage.3 The pyrrolidine dithiocarbamate not only can reduce blood glucose levels, but also decrease the diabetic pancreatic isletβcell apoptosis by relieving the oxidative stress reaction of diabetic rats through suppring the expression of iNOSand NT production.