The Regulatory Mechanisms Of C-peptide On NF-?B And P300 To Prevent And Cure Diabetic Nephropathy

Objective:Diabetes mellitus(DM)is the third non-infectious chronic disease following cancer and cardiovascular disease,seriously threatening human health.It is reported that 382 million people are suffering from DM worldwide,and it will rise to 592 million by 2035.Nearly 100 million people suffer from diabetes in China,which is equivalently around a quarter of the global diabetic patients.Diabetic nephropathy(DN)is one of the most common diabetic microvascular complications.It is a leading cause of end-stage renal disease and a high risk of mortality and morbidity.According to epidemiology,approximately 35%?50%type 1 and 20%type 2 DM patients will develop nephropathy.The major pathological change of DN is glomerulopathy,including thickening of glomerular basement membrane,accumulation of extracellular matrix and eventually leading to glomerulosclerosis.To date,the mechanisms of DN have not been fully elucidated.As all know,oxidative stress is the most pivotal metabolic consequence in the development and progression of DN.The key enzyme of oxidative stress is inducible nitric oxide synthase(iNOS).In RAGE/iNOS transgenic mice,30?40%of glomeruli exhibited nodule-like lesions.As the capital regulatory factor of iNOS,nuclear factor-kappa B(NF-?B)would translocate into the nucleus to regulate the transcriptions of target genes when activated.As the most important co-activator,P300 participates in the regulation of multiple transcription factors with the activity of nucleoprotein acetyltransferase.The binding of NF-?B P65 and the co-activator P300 activated the transcriptions of target genes abnormally and increased iNOS produced,which played a vital role in the development and progression of DN.C-peptide is a 31 amino-acid polypeptide generated in the biosynthesis of insulin and secreted in equimolar concentrations with insulin.Over the years,it has only been evaluated as an indicator of pancreatic ?-cell function.Numerous researches in both patients and animals with DM have demonstrated that exogenous C-peptide might play an important role in preventing and potentially reversing DN.Furthermore,a series of studies have discovered that physiological concentrations of C-peptide could play cytoprotective effects by binding to G-protein-coupled receptors of membrane or internalizing into the cytoplasm or nucleus.Our preliminary study has demonstrated that 0.5 nmol/L human C-peptide located in the nucleus of rat glomerular mesangial cell line(HBZY-1)stimulated with high glucose(25 mM).C-peptide could inhibit the expression of iNOS and the nuclear translocation of NF-?B in HBZY-1 cells exposed to high-glucose.Nevertheless,it is still unclear whether C-peptide prevents DN by regulating NF-?B and P300.This study was envolved in vivo and in vitro.Rat glomerular mesangial cell line(HBZY-1)was routinely cultured.A laser scanning confocal microscope(Confocal)was used to observe the effects of 0.5 nM human C-peptide on NF-?B and the co-localization of NF-?B and P300 in cells stimulated with high glucose(25 mM).The regulatory effect of C-peptide on NF-?B activating iNOS gene was detected by means of chromatin immunoprecipitation(ChIP)assay.Besides,the effect of C-peptide on the interaction of NF-?B and P300 was assessed by co-immunoprecipitation(Co-IP)assay.On the other hand,healthy male Sprague-Dawley(SD)rats were induced with streptozotocin(STZ)into diabetic model.All rats were randomly divided into normal control group(NC),diabetic group(DM),diabetic prevented with human C-peptide group(DM+CP-P),diabetic treated with C-peptide group(DM+CP-T)and diabetic treated with scrambled C-peptide group(DM+scCP).Blood glucose and body weight of all rats were measured before and after the experiment,respectively.The 24-hour urinary protein excretion was also determined.Moreover,the morphological changes of the renal cortex in all groups were observed under optical and transmission electron microscopy(TEM),in order to explore the efficacies and effects of C-peptide on prevention and treatment of DN.Method:1 Cell Experiments1.1 Cell cultureRat glomerular mesangial cell line(GMCs)HBZY-1 were maintained with Dulbecco's modified Eagle's medium(DMEM)containing 5.5 mmol/L D-glucose which is considered the normal glucose level and 10%fetal bovine serum(FBS)at 37? in a humidified incubator with 5%C02.0.25%EDTA-trypsin was used to digest and inoculate in 100 ml flasks.1.2 Experimental design and groups(1)NF-?B P65 nuclear translocation and its co-localization with P300 were observed by Confocal,to determine the best time for C-peptide effects.The experiment was divided into the following five groups:1)normal control group(Control),GMCs(HBZY-1)were maintained with DMEM contained 5.5 mmol/L D-glucose,which is considered the normal glucose level;2)hyperosmotic control group(HO):GMCs were stimulated with DMEM contained 25 mmol/L L-glucose for 24 hours;3)high glucose group(HG):GMCs were stimulated with DMEM contained 25 mmol/L D-glucose for 24 hours;4)NF-?B inhibitor(BAY11-7082)plus high D-glucose DMEM group(IH):GMCs were cultured with normal DMEM which added 10 ?mol/L BAY11-7082 for 1 hour,and then exchanged high D-glucose DMEM for 24 hours;5)C-peptide treatment group(CP):GMCs stimulated with high D-glucose DMEM for 24 hours were treated with DMEM contained 0.5nM C-peptide and high D-glucose for 10,20,30,40,50 and 60 minutes,respectively.(2)ChIP assay was performed to detect the regulatory effects of C-peptide on GMCs NF-?B activating iNOS gene.It was divided into three groups below:1)normal control group(Control),GMCs(HBZY-1)were cultured with normal DMEM;2)high glucose group(HG):GMCs were stimulated with high D-glucose DMEM for 24 hours;3)C-peptide treatment group(CP):GMCs were treated with DMEM contained 0.5 nM C-peptide and high D-glucose for 30 min after stimulating with high glucose DMEM for 24 hours.(3)Co-IP assay was used to detect the effects of C-peptide on the interaction between NF-?B P65 and P300 in GMCs.The experiment was divided into the following three groups:1)normal control group(Control),GMCs(HBZY-1)were cultured with normal DMEM;2)high-glucose group(HG):GMCs were stimulated with high D-glucose DMEM for 24 hours;3)C-peptide treatment group(CP):GMCs were treated with DMEM contained 0.5nM C-peptide and high D-glucose for 30 min after stimulating with high D-glucose DMEM for 24 hours.2 Animal Experiments2.1 Experimental design and groups80 healthy male SD rats were randomly divided into two groups:the normal control group(NC,n=8),and the remaining 72 rats received a single intraperitoneal injection of 45 mg/kg STZ(10 mg/ml,freshly dissolved in 0.1 mol/L citrate buffer,pH 4.4)to induce diabetic model.Three days later,blood glucose from tail vein was measured.If the levels of blood glucose were not less than 16.7 mmol/L and urine glucose(+++)or above,the diabetic models were successful.68 successful diabetic models were randomly divided into 4 groups:diabetic group(DM,n=17),diabetic prevented with C-peptide group(DM+CP-P,n=17),diabetic treated with C-peptide group(DM+CP-T,n=17)and diabetic treated with scrambled C-peptide group(DM+scCP,n=17).The above five groups were fed on a normal diet in the standard environment(20-25?).Rats in the DM+CP-P group were received subcutaneous injection of 130nmol/kg human C-peptide twice daily for 6 weeks(starting at onset of diabetes).Rats in the DM+CP-T and DM+scCP groups were received the same dosage and method of human C-peptide or scrambled C-peptide for 6 weeks(starting 6 weeks after onset of diabetes).After the experiment,all rats were sacrificed by femoral artery bleeding.2.2 blood glucose and 24-hour urineBlood was obtained from the tail vein to measure the concentrations of blood glucose by glucometer before STZ injection as well as 3 days and 12 weeks after STZ injection.24-hour urine was collected for determination of 24-hour urinary protein excretion.2.3 Morphological observationRenal cortex was taken for biopsy and observed under optical microscope and TEM.Results:1 The effects of C-peptide on NF-?B P65 nuclear translocation and its co-localization with P300 in GMCs at different timesConfocal results have showed that NF-?B P65 was induced nuclear translocation,and co-localized with P300 in the nucleus after high glucose stimulated GMCs for 24 hours.0.5 nM C-peptide treated for 30 minutes,the effects were significant,that was,NF-?B P65 was scattered in the cytoplasm and P300 was scattered in the nucleus.At 40?60 minutes,C-peptide effects gradually decreased and almost disappeared.Therefore,we selected 30 minutes as the best time for C-peptide effects on high glucose stimulated GMCs.2 ChIP assay was performed to detect the regulatory effects of C-peptide treatment for 30 minutes on NF-?B activating iNOS in CP groupAfter GMCs were stimulated with high glucose for 24 hours,the binding of NF-?B and the proximal promoter region of iNOS in HG group(4.44 ± 0.29,P<0.05)was significantly higher than that in Control group.However,0.5 nM C-peptide treated for 30 minutes,the binding of NF-?B and the proximal promoter region of iNOS in CP group(1.86 ± 0.21,P<0.01)obviously decreased compared with the HG group.After GMCs were stimulated with high glucose for 24 hours,the binding of NF-?B and the distal promoter region of iNOS in HG group(5.06 ± 0.43,P<0.05)was significantly higher than that in Control group.Next,0.5nM C-peptide treated those cells for 30 minutes,the binding of NF-?B and the distal promoter region of iNOS in CP group(0.94±0.07,P<0.05)significantly reduced compared with the HG group.3 Co-IP assay was performed to evaluate the effects of C-peptide treatment for 30 minutes on the interaction between NF-?B P65 and P300 in GMCs of Control,HG and CP groupsThe cell protein lysates of the Control,HG and CP group were all added to P300 antibody for precipitation,and the binding of NF-?B P65 and P300 was analyzed by Co-IP assay.Western blot showed that the binding content of co-immunoprecipitated NF-?B P65 and P300 in GMCs of HG group(18.31±0.82,P<0.05)were significantly higher than that in Control group(5.99±0.07).In contrast,the binding content of co-immunoprecipitated NF-?B P65 and P300 in CP group(5.220± 0.33,P<0.01)significantly decreased compared with the HG group.The cell protein lysates of Control,HG and CP group were all added to P65 antibody for precipitation,and the binding of P300 and NF-?B P65 was analyzed by Co-IP assays.Western blot showed that the binding content of co-immunoprecipitated P300 and NF-?B P65 in GMCs of HG group(9.19 ±0.17,P<0.01)were significantly higher than that in Control group(3.54 ±0.21).By comparison,the binding content of co-immunoprecipitated P300 and NF-?B P65 in CP group(2.98± 0.23,P<0.01)significantly declined compared with the HG group.4 General symptoms and the effects of C-peptide on blood glucose and body weight in DM ratsThe successful diabetic models gradually appeared several symptoms like weight loss,loose fur without politure,clumsy reaction,polydipsia,polyuria,polyphagia and growth retardation and so on.Along with the experiment,the symptoms of rats in DM and DM+scCP-T groups obviously worse,while the symptoms in DM+CP-P,DM+CP-T groups were similar with the NC group.Rats selected in all groups have no significant difference in blood glucose and body weight.No significant difference(P>0.05)in blood glucose was observed among DM+CP-P group which average blood glucose was(29.96±0.52)mmol/L,DM+CP-T group(28.90 ± 0.74)mmol/L,DM+scCP-T group(29.74 ± 0.66)mmol/L and DM group(29.23 ± 0.66)mmol/L throughout the experiment.But the levels of average blood glucose in above four groups were significantly higher than that in NC group(6.40±0.25)mmol/L(P<0.001).By the end of C-peptide treatment,there were no significant difference(P>0.05)among DM+CP-T group with the average weight(271.82± 8.76)g,DM+scCP-T group(248.35 ± 7.19)g and DM group(249.0 ± 7.13)g,while the average body weight in DM+CP-P group(368.06 ±10.09)g had obviously significant difference(P<0.001)from DM group.Nevertheless,body weights in above four groups significantly decreased compared with the NC group(515.5 ± 14.42)g(P<0.001).At the end of C-peptide treatment,the DM group with the 24-hour urinary protein excretion(327.93 ± 32.58)mg and the DM+scCP group(293.79 ± 49.40)mg were significantly higher than the NC group(15.42 ±4.06)mg(P<0.05);DM+CP-P group(55.21± 4.06)mg and DM+CP-T group(70.2±9.46)mg group was significantly lower than that in DM group(P<0.05).5 The observation of the glomerular morphologyObservation of glomeruli under optical microscopy(×400):normal glomeruli was found in NC group;the DM group was similar with the DM+scCP group,abnormal glomerular and renal cysts increase were observed,and there was glomerular necrosis in DM+scCP group;whereas the renal capsular space shrinking was showed in DM+CP-P and DM+CP-T group improved compared with the DM group,and the lesions in the DM+CP-P group were less than that in DM+CP-T group.Observation of glomeruli under TEM(×20K):normal glomeruli was observed in NC group;the DM group showed that the basement membrane severely thickened with local hummocky eminence,vascular endothelial cells were severe proliferation,and podocyte cells and foot process mostly fused,which were similar with the DM+scCP group;in DM+CP-P and DM+CP-T groups,the basement membrane and vascular endothelium were nearly normal,and podocyte cells and foot process mildly fused,significant alleviated compared with the DM group.Moreover,the pathological changes in DM+CP-P group were better than those in DM+CP-T group.Conclusion:1 The regulatory mechanisms of NF-?B and the co-activator P300 by C-peptide on prevention and treatment of diabetic nephropathy are:(1)C-peptide could inhibit NF-?B P65 nuclear translocation and the co-localization of NF-?B P65 and P300.(2)C-peptide could inhibite the obvious interactions between nuclear translocated NF-?B and the proximal or distal promoter region of iNOS in GMCs after they were stimulated with high-glucose for 24 hours,and significantly inhibited iNOS gene transcription activated by NF-?B.(3)C-peptide could inhibite the interaction between NF-?B and P300 in high-glucose stimulated GMCs(HBZY-1).2 According to optical microscopy and TEM,C-peptide can significantly improve the functional and morphological changes of glomeruli in DM rats.