Protective Effects Of Pyrrolidine Dithiocarbamate On Islet β Cell Oxidative Injury In Type2Diabetic Rats

Diabetes mellitus has become one of the major chronic diseases in the21st century, which threats human health. With the rapid development of social economy, the improvement of living level and the change of the life style, the probability of suffering diabetes is increasing rapidly. The explosion of T2DM patients is a major reason of leading to increasing of the aggregate number of diabetes in the world. The number of Chinese T2DM patients is the second place in the world. The defects of pancreatic β cells and insulin resistance are the pathological basis of type2diabetes. The present study shows that the hyperglycemia and hyperlipemia in vivo of patients with type2diabetes induce oxidation and antioxidation disording and resulting oxidative stress, which is a major reason to cause the impairation of islet (3cells and diabetes progression. High level of blood glucose can make the cells produce excess reactive oxygen species (ROS), which induce oxidative stress and activate NF-κB signing pathway and increase the amount of nitric oxide (NO) by the inducible transcription of nitric oxide synthase (iNOS). NO can induce β cells apoptosis by many ways. In addition, excessive NO can bind with superoxide anion (O2) to generate peroxynitrite (ONOO"), which is a more potent oxidant, impair and facilitate (3cells apoptosis. ONOO" makes the tyrosine residues of protein nitration, finally producing nitrotyrosine (NT) Protein nitration can induce the loss of enzyme activity, cell necrosis and apoptosis.Pyrrolididne dithiocarbamate (PDTC) is a dithiocarbamates, which is a thiol-containing compound that can chelate various metal ions and pyrrolididne dithiocarbamate has the function of anti-oxidant. Pyrrolididne dithiocarbamate can specificly suppress nuclear factor-KB (NF-κB) activity, and pyrrolididne dithiocarbamate efficiency inhibits the oxidative damage. Type2diabetic rat model were established by small doses streptozotocin (STZ) and high-fat diet. Interveneding with pyrrolidine dithiocarbamate (50mg/kg/d,IP) for a week, then blood glucose and blood lipid were evaluated; superoxide diamutase (SOD), glutathione, peroxidase (GSH-PX) activity, and malonaldehyde (MDA) content in pancreatic tissues were examined, iNOS expression and NT production in pancreatic tissues were detected and the apoptosis rate of pancreatic islet β cells was measured, and certained the effects of pyrrolidine dithiocarbamate on againsting oxidative stress and nitrification to protect islet (3cells from impairing. To measure the expression level of transcription factor PDX-1, Ac-FoxO1, P-FoxO1and the content of insulin and the proliferation of β cells, discussing the possible mechanism about the PDTC can promote insulin secretion in pancreatic islet β cells. These experiments consist of the following three parts: Part one Pyrrolidine Dithiocarbamate improves insulin resistance of T2DM ratObjective:Through high-fat diet and a low dose streptozotocin (STZ) via intraperitoneal injection to establish the rat model of type2diabetes. To explore influence of Pyrrolidine Dithiocarbamate improving insulin resistance and insulin sensitivity of T2DM rat.Methods:37healthy180-210g, age of8-week-old male Wistar rats were purchased from Hebei Medical University Animal Centre, the rats were divided randomly into2groups:the normal control group (NC group) received regular diet and the high-fat diet group (HFD group) received high-fat diet for8weeks. When the high-fat diet animals appeared insulin resistance, evaluated by oral glucose tolerance test (OGTT), STZ was administered via intraperitoneal injection at27mg/kg to make model. Control group administers the same volume citrate buffer solution, and each group rats were fed with original forage.After72h, diabetes mellitus rats were confirmed by the random glucose over16.7mmol/L. Then the diabetic rats were further divided randomly into2groups:T2DM group (DM group) rats were fed with high-fat diet continuously for a week. PDTC-treated group (DM+PDTC group) rats were fed with high-fat diet and administered with PDTC via intraperitoneal injection at50mg/kg once daily. Rats in NC and DM groups were given the same volume of saline. There were12rats in NC group,12rats in DM group,12rats in DM group,12rats in PDTC group at the end of experiment.Result:1. Establishment of insulin resistant model in high-fat diet ratsAfter8weeks, the blood glucose levels of the HFD group in every time were higher than that of the NC group and the peak times were delayed, the blood glucose levels reached maximum at90min and failed to decline to the control levels at120min, at the time of90min,120min the blood glucose levels were significantly higher than in the NC group (P<0.05).The mean values of the AUCglucose in the HFD group were significantly higher than in the NC group (P<0.05). After glucose ingestion, the insulin levels of the HFD in every time was higher than in the NC group (P<0.01).The mean values of the AUCinsulin in the HFD group were significantly higher than in the NC group (P <0.01). The result of ITT:the HFD-fed rats showed higher glycemia after insulin administration compared with the control rats, and the ITT AUC was significantly larger in the HFD group (P<0.01). In a word, rats in the HFD group appear insulin resistance2. PDTC can improve insulin resistance of T2DM ratsAfter treatment with PDTC, the blood glucose levels and insulin levels were lower than the DM group. Then declined to the control levels by120min in PDTC-treated DM group, at the time of120min the blood glucose was lower than in DM group(P<0.05). The mean values of the AUCglucose were found to not be significantly between the two experiment groups. After glucose ingestion, the insulin levels of PDTC-treated DM group in every time was lower than in DM group (P<0.01). The mean values of the AUCinsuiin were found to not be significantly between the two experiment groups. The result of ITT:the HFD-fed rats showed higher glycemia after insulin administration compared with control rats, and the ITT AUC was significantly larger in the HFD group (P<0.01).Conclusions:1. The pyrrrolidine dithiocarbamate (PDTC) can reduce blood glucose levels of the diabetic rats.2. Pyrrolidine Dithiocarbamate improves insulin resistance and insulin sensitivity of T2DM rat Part two Production effects of PDTC on islet β cell oxidative damage in Type2diabetes ratObjective:To assay the expression levels of iNOS, the production of the NT and the apoptosis rate of pancreatic islet β cell, investigating the effects and mechanism of protecting islet (3cell from impairing.Method:Thought the method of the immunohistochemitry, immunofluorescence and Western blot detected the inducible transcription of the nitric oxidative syntheses (iNOS)、and the protein nitration (NT) production and observe both in nucleus and cytoplasm of NF-κB in the pancreatic. The paraffin sections of each group were made by the routine method and proceed with HE staining to observe general appearance of the pancreatic islet. And thought the method of flow cytometry assay of the apoptosis rate of pancr eatic islet cell.Result:1. Comparison of the rate of the pancreatic islet P-cell apoptosis in each group:After the treatment with the PDTC the rate of the pancreatic islet β-cell apoptosis was significantly lower than in the DM group(P<0.05).2. The morphplogical changes of the pancreatic islet cell:Under light microscope, HE-staining showed that pancreatic islet structure was clear, the pancreatic islet cells linked tight, fairly uniform, the cell nucleus were stained violet-blue, giant round, the cell nucleus were plenty of cytoplasm in the NC group. In the DM group the structure of pancreatic islet was not clear, the number of pancreatic islet cells were decreased, and some islet cells became vacuole. In the PDTC group the number of the pancreatic islet cells was larger than those of the DM group, and the structure of the pancreatic islet was obviously improved.3. Expression of the iNOS in the pancreatic tissue in the different groups:Immunohistochemical staining results of the iNOS showed the pos-itive signal were brown in color, there had faint iNOS positive signal in the pancreatic tissues of the NC group, and after administration PDTC, the positive signal of the iNOS was significantly fainter than the DM group(.P<0.01); Western-blotting analysis of the iNOS expression showed the expression of the iNOS in the DM group rats’pancreas was significantly higher than in the NC group. After administration PDTC, the expression of the iNOS in pancreas was significantly lower than in the DM group.4. Expression of NT in pancreatic tissue in the different groupsImmunohistochemical staining results of NT showed the positive signal were brown in color, there had faint NT positive signal in pancreatic tissues of the NC group, and after administration PDTC, the positive signal of the NT was significantly fainter than the DM group(P<0.01); Western-blotting analysis of the NT expression showed the expression of NT in the DM group rats’pancreas was significantly higher than in the NC group. After administration PDTC, the expression of the NT in pancreas was significantly lower than in the DM group.5. Observing the NF-κB expression both in nucleus and cytoplasm in the pancreatic islet (3cells in the different groupsWestern-blotting analysis of the NF-κB expression showed the expression of NF-κB in the nucleus of the DM group rats’pancreatic islet β cells was significantly higher than in the NC group. After administration PDTC, the expression of NF-κB in nucleus of pancreatic islet β cells was significantly lower than in the DM group.Conclusions:1. Impairment of the pancreatic islet defense from the oxidative damage in the type2diabetic rats, imbalance of redox, meanwhile over expression of the iNOS and the NT in the pancreatic islet demonstrate that oxidative stress play an important role in pancreas damage of the diabetes.2. The Pyrrolidine Dithiocarbamate not only can reduce blood glucose levels, but also can attenuate production of the iNOS, the ONOO-and the NT in pancreatic islet and delay pancreas oxidative damage in the diabetic rats, thus decreased the diabetic pancreatic islet β cells apoptosis. So early application of the PDTC can prevent pancreas damage of the diabetes. Part3Pyrrolidine Dithiocarbamate affects islet β cell synthesis by regulating transcriptional factor PDX-1and FoxO1Objective:After the PDTC treatment, through the assessment of the content of the insulin, and the expression levels of the transcription factors PDX-1, Ac-FoxO1and p-FoxO1in the pancreatic islet of each group rat model, exploring the possibly molecule mechanism of the PDTC protection for the pancreatic islet β cells function and amount.Method:Thought the method of the immunohistochemitry we can assay the content of the insulin in each group. And through the Western blot and immunohistological methods we can detect the expression levels of the PDX-1, the Ac-FoxO1and the P-FoxO1in the pancreatic islet β cells.Result:1. Immunohistochemical staining results of insulin:The positive signal insulin was fainter in pancreatic tissues of DM group, and after administration PDTC the positive signal of insulin was significantly higher than DM group;2. Expression of PDX-1in pancreatic tissue of different group and its transcription between cytoplasm and nuclear in pancreatic islet β cells:Immunohistochemical staining results of PDX-1showed the positive signal were brown in color, the positive signal of PDX-1was fainter in pancreatic tissues of DM group, and after administration PDTC the positive signal of PDX-1was significantly higher than the DM group; Immunofluorescence chemical staining results of PDX-1showed the positive PDX-1expressed in nucleus of NC group, expressed faint PDX-1positive signal in DM group, after administration PDTC, the positive signal of PDX-1in nucleus of pancreatic β cells was significantly higher than in DM group; Western-blotting analysis of PDX-1expression in each group showed the expression of the PDX-1in nucleus of the DM group rats were lower than the NC group, after administration PDTC, the expression PDX-1in nucleus of pancreas were significantly higher than the DM group; The expression of PDX-1in cytoplasm of DM group were significantly higher than the NC group, after administration PDTC, the expression PDX-1in cytoplasm of pancreas were significantly lower than the DM group.3. Expression of the p-FoxO1in the pancreatic tissue of different group and its transcription between cytoplasm and nucleoluse in pancreatic islet (3cellsImmunohistochemical staining results of p-FoxO1, showed the positive signal were brown, there had faint p-FoxO1positive signal in nucleus of pancreatic tissues in NC group, and after administration PDTC, the positive signal of p-FoxO1was significantly fainter than DM group; Immunofluorescence chemical staining results of p-FoxO1showed the positive p-FoxO1expressed in the green fluorescen, the expression of p-FoxO1in nucleus of DM group were significantly higher than the NC group, and after administration PDTC, the expression of p-FoxO1in nucleus of pancreatic (3cells were significantly lower than the NC group.4. Expression of the Ac-FoxOl in the pancreatic tissue of different groupWestern-blotting analysis of Ac-FoxO1expression in each groupshowed the expression of the Ac-FoxOl in DM group rats’pancreas was significantly higher than in NC group, after administration PDTC, the expression of Ac-FoxOl in pancreas was significantly lower than in DM group.Conclusions:PDTC could inhibite phosphorylation and acetylation of FoxO1, PDTC prevent p-FoxO1from its nuclear import, which solved the inhibition of PDX-1, PDTC promoted the synthesis insulin of pancreatic islet β cells.