PARP Is Involved In Thedevelopment Of Peripheral Diabetic Neuropathy Via Regulation OfNF-κB

Objective:To establish animal model of peripheral neuropathy of 2-type diabetes mellitus rat and observe its characteristics and experimental application value, to provide a reliable experimental basis for researching the pathogenesis of peripheral neuropathy of 2-type diabetes mellitus rat; to observe change of PARP and NF-κB, when primitive DPN rat is in 3-AB, to observe the change of PARP and NF-κB and the relations between them, when PC-12 cell is in high glucose environment and PARR inhibitor 3-aminobenzamide;Method:1. Animal model:80 8-week-old Sprague-Dawley male rats,170-190g, after adaptive feeding in a week, they are randomly divided into two groups,30 in normal control group,50 in the experimental group. In the normal group, they are fed with normal diet, in the experimental groups, they are fed with high-fat high-sugar diet, after 4 weeks, in the experimental group, they are first injected 1% streptozotocin (STZ,25mg/kg) to abdominal cavity, in the normal group, they are injected 0.1 mmol/L citric acid-sodium citrate buffer with the same volume. After 4 weeks,1% streptozotocin (STZ,35mg/kg) is injected to the abdominal cavity for the second time, weight and blood glucose of rats at different times are detected, the rats with random blood glucose>16.7mmol/L are 2-type diabetes model, the rats with less than 15.0mmol/L blood sugar level are excluded, peripheral blood is taken, glucose, blood lipids, glycated hemoglobin are measured by Olympus automatic biochemical analyzer.2. In vivo experiments:Using immunity coprecipitation method to discuss NF-κB p50 and p65, selecting appropriate NF-κB as detection target.The experiment is divided into three groups:(1) control group (normal rats) (2) DPN group (rats suffering from DPN) (3) DPN+3-AB treatment group (regular daily gavage 30mg/kg 3-AB). The sciatic nerves were analyzed using electron microscopy. As observed in the ultramicrostructures of the ischium of male SD rats, in DPN group the myelin sheath cells became disordered in the medullated fibers of sciatic nerves, and shrinkage, deformation and inhomogeneity can be observed in neuritis. The structures of the Schwann cells blurred and the vacuoles degenerated. Cells of non-myelinated nerves swelled and blebbed, and morphological irregularity and widened cellular spaces were observed. Additionally, the Schwan cells shrank. The pathological structures of neuritis and myelin were significantly improved after treatment with3-AB, an PARP inhibition. Deranged myelin sheath cells recovered and Schwann cell swelling was attenuated.Using flow cytometry to observe death rate of ischiadic nerve cells of above groups, respectively using PT-PCR and western blot method to detect mRNA and protein level change of PARP and NF-κB in rat sciatic nerves.3.Experiment in vitro:first, the experiments are divided into three groups:(1) control group (cultivating in DMEM low glucose medium) (2) HG group (cultivating in DMEM high glucose medium) (3) HG+3AB group (cultivating in DMEM high glucose medium containing 0.25mmol/L 3-AB). Using flow cytometry to observe the death rate of the above PC-12 cells of each group, respectively using PT-PCR and western blot method to detect mRNA and protein level change of PARP and NF-κB in cell.Results:1. Animal model:compared with 483.65±14.90g of normal group, the 12 week after STZ-induced, the weight of the model group is 265.76±46.76g, which has statistical significance (P<0.01), blood lipid test of rats of model group:TG level is 1.90±0.23; TC level is 1.76±0.28; LDL level is 0.88±0.27, compared with the normal group, which has statistical significance (P<0.01); glycated hemoglobin level is 6.97±0.18, compared with the normal group, which has statistical significance (P <0.01). Insulin level measurement of rats of test group:insulin level is 39.32±+2.60, compared with the normal group, which has statistical significance (P<0.01); insulin resistance index is 41.92 ±5.35, compared with the normal group, which has statistical significance (P<0.01); 3. The nerve muscle electricity physiology parameters of rats in experimental group:sciatic nerve conduction latency is 1.02± 0.32, compared with the normal group,1.47+0.12, which has statistical significance (P<0.01).2. In vivo experiment:the mutual influence of NF-κB p50 and PARP is greater than NF-κcBp65, flow cytometry shows that high glucose environment can disrupt the PC-12 cell cycle, flow cytometer detection apoptosis rate result control group apoptosis rate (%) is 3.59±0.31. Compare with it, the obvious increase of DPN group sciatic nerve apoptosis rate(%) is 33.44 ± 1.72, the difference of two group apoptosis rate has statistical significance (P<0.01). The apoptosis rate of DPN+3-AB group is 15.55 ± 1.03, and its apoptosis rate has significant differences to DPN group. RT-PCR relative quantitative results show that:the value of PARP mRNA and NF-κB mRNA P of control group, DPN group, DPN+3-AB group is 0.000, indicating that P<0.05, the difference has statistical significance, that is, PARP mRNA and NF-κB mRNA P of the three group are incompletely same, at least, two groups are not equal, comparing among groups, control group comparing with DPN group, and DPN+ 3-AB group, P<0.01, difference has statistical significance, DPN group comparing with control group, DPN+3-AB group, P<0.01, difference has statistical significance, DPN+3-AB group comparing with control group, DPN group, P<0.01, difference has statistical significance. Western blot experiment results show that:P value of PARP protein level and NF-κB protein level of control group, DPN group, DPN+3-AB group is 0.000, indicating that P<0.05, the difference has statistical significance, that is, PARP protein level and NF-κB protein level are not the same, at least, two groups are not equal, comparing among groups, control group comparing with DPN group, and DPN+3-AB group, P<0.01, difference has statistical significance, DPN group comparing with control group, DPN+3-AB group, P<0.01, difference has statistical significance, DPN+3-AB group comparing with control group, DPN group, P<0.01, difference has statistical significance.3. Experiments in vitro, the cell mortality rate increases, and the cell mortality rate is little in the high glucose solutions with PARP/NF-κB inhibitor. RT PCR and western blot display that, mRNA and protein levels of PARP and NF-κB go up in high glucose environment compared with low-sugar environment, the contents of PARP in the high glucose solutions with 3-AB declines compared with the high glucose solutions without inhibitor.Conclusion:the experimental 2-type diabetic neuropathy rat model with insulin resistance features and similar to human can be duplicated by one-week high-sugar-fat-diet combined with twice low dosage STZ injection. The model has the characteristics of peripheral neuropathy, is an ideal animal model for the study of pathogenetic mechanism of diabetic peripheral neuropathy. The model has good stability after modeling, and is conducive to do study of related influencing factors of the drug intervention. In vivo experiments show that the early application of PARP inhibitors can improve nerve conduction velocity of early neuropathy of 2-type diabetes, provide experimental evidence for diabetic neuropathy, and provide drug choice basis for treatment of diabetic neuropathy; in vitro experiments show that short-term application of PARP inhibitor can reduce death rate of cell in high glucose environment, activated PARP can upregulate the expression of factors, PARP regulates activity of transcription factor.