The Relationship Of Dnase I With Pancreas Injuries By High Glucose In Type2Diabets:from BC1-2/Caspase-3Pathway To Explore The Relationship Of Dnase I With The Injuries Of Pancreas

Type2diabetes mellitus is a complex metabolic disorder that afflicts hundreds of millions of people worldwide..It is estimated that there are about7million people diagnosed with diabetes each year in the world and a person dies of diabetes-related diseases every10seconds. However, the underlying mechanisms have not been elucidated. DNase I is a38-kDa glycoprotein. As one kind of Ca2+/Mg2+dependent nonrestriction nucleases, it can hydrolyze phosphodiester bonds both in single and double stranded DNA to generate single nucleotides and oligo nucleotides with5’-phospho and3’-hydroxy termini. It is physiologically a benign extracellular enzyme present ubiquitously in blood serum and body secretions. It is responsible for the degradation of the chromatin released by dead cells, which have not undergone apoptosis. In the process of cell apoptosis, the DNA of dying cells’can be degraded into180-200bp fragments and finally is completely degraded into nucleotides to participate in the new DNA replication.At present, there are two diseases-SLE (systemic lupus erythematosus) and AMI (acute myocardial infarction) known to be associated with increased levels of DNase I in serum. However, as an enzyme that is secreted by pancreas, the underline mechanisms of it with type2diabets are still in vain. Human pancreas exhibits the higher DNase I activity. It was reported that60-65%of serum DNase I was secreted by pancreas. It is still not clear whether there is some correlation between the DNase I and the diabetes. In this study, we want to explore the relationship between DNase I and T2DM using the sera and pancreas tissue both in human and rat.Objective:By using of clinical samples, animal samples and cells, we explore the relationship of DNase I increase with pancreas injuries by high glucose in type2diabetes. Through the study we hope to supply a new thought and pathogenesis reasons fot the investigation of pancreas injuries in type2diabetes. Meantime, we can provide a new molecular diagnosis marker of pancreatic injury and scientific basis for gene therapy. Methods:(1) To examine the DNase I activity in serum in patients with type2diabetes.To examine the DNase I activity in serum in patients with type2diabetes, we collected serum of patients and healthy volunteers in our hospital. The serum enzyme activity was detected by radial enzyme-diffusion method.(2) in vivo studies of DNasae I expression in pancreas.This part of the experiment mainly divided into two parts. The first part was done by collecting the the pancreas of patients with pancreatic cancer, to observe the locations of DNase I in the pancreas in human. The second part mainly through observation type2diabetes rats induced by high-fat food plus low dosage STZ injection to explore the protein expression of DNase I, Caspase-3and Bcl-2in diabetes. In this part of the experiment immunohistochemistry, Western Blot and Real-time PCR was used for the detection.(3) High glucose stimulated the beta cells proliferation and apoptosis.INS-1cells were simulated by30mmol/L glucose concentration to observe the changes of DNase I under glucose environment. Meantime, DNase I siRNA was also used to observe the Caspase-3and Bcl-2changes in the environment of DNase I knocked down. In addition, we gave the INS-1cells exogenous DNase I to explore the overexpression of DNasae I to the levels of Caspase-3and Bcl-2.(4) DNase I expression in kidneyTo observe the relationship of DNase I with diabetic nephropathy, we had observed the DNase I, Bcl-2and Caspase-3expression in kidney of type2diabetic rats.Results:(1) Compared with healthy people, the activity of serum DNase I in patients with type2diabetes showed a significantly increased (p<0.01). And this increase seems do not affected by gender. At the same time in rats serum, we also found that the activity in2weeks and20weeks had both significantly increased in diabetic group (p<0.05)(2) Immunohistochemistry results of both human and rats showed that DNase I was mainly expressed in islets. However, in type2diabetes the DNase I expressions in islet were increased. And high glucose can induced the DNase I expression in acinus increased significantly (p<0.001). Western Blot results showed that the protein level of DNase I was significantly increased in rats pancreas both in2weeks and20weeks (p<0.05). However, compared with normal group, the Bcl-2in diabetic rats in the second week dropped significantly (p<0.05), while in20weeks, no significant difference was observed. In addition, there was no significant difference of caspase in both2weeks and20weeks.(3) Cell experiments have shown that DNase Ⅰ was widely expressed in cytoplasm and nucleus. High glucose can induced DNase Ⅰ and Caspase3expression significantly increased in INS-1cells (p<0.05). Meantime, we had used DNase Ⅰ siRNA to knock down the DNase Ⅰ expression, and we had found that once DNase Ⅰ was knocked down, the cell apoptosis rate and Caspase-3level can be greatly decreased even in high glucose simulated group (p<0.05). Flow cytometry results showed that the cell apoptosis rate in Normal group was only7.9%, the high glucose group was18.1%, whereas the apoptosis rate in the knocked down group was only9.9%.(4) INS-1cells in normal group was given5mU/ul and50mU/ul of DNase Ⅰ, then we had observed the apoptosis rate was only7.2%in normal group, and6.9%in5mU/ul DNase Ⅰ group and9.8%in50mU/ul DNase Ⅰ group. In addition, we had foun that the apoptosis rate in high glucose group was17.3%,21.8%with5mU/ul DNase Ⅰ group and34.6%in50mU/ul DNase Ⅰ group.(5) Electron microscopy results showed that at20weeks, rat kidney basement membrane and podocyte can be observed with serious damage; PAS results showed that there were obviously glycogen depositions in the glomerular in diabetic rats. Further analysis found that there was a significantly increase of DNase Ⅰ expression in both2weeks and20weeks of rat kidney (p<0.05).Conclusion:(1) Patients with type2diabets showed a significantly increase of DNase Ⅰ activity in serum, while this increase was also observed in type2diabetic rats, we speculate that the DNase Ⅰ may participate in the process of diabetes and it may involved in the injuries of islets and acinus induced by high glucose.(2) High glucose stimulation can lead to DNase Ⅰ expression increased, at the same time, cell apoptosis rate were also increased significantly. However, knowed down of DNase Ⅰ can significantly alleviate the cell apoptosis rate. Base on the results, we speculated that DNase Ⅰ may participate in the process of pancreas and kidney damage caused by high glucose.This is the first study that had discoveryed the DNase Ⅰ activity was increased in serum of type2diabetic patients. Meantime, for the first time we also found that high glucose can induced the significant increase of DNase Ⅰ in pancreatic acinus; through the in vitro study, we also proofed that DNase Ⅰ increase can promote β cells apoptosis under high glucose condition. By doing this researches we can supply a new thought for the mechanism study of type2diabetes.