Role Of Proteasome Inhibitor MG132 On ERK1/2 Pathway In The Early Diabetic Nephropathy Rats Model

Abstract:Objective:Diabetic nephropathy(DN) is one of the most devastating microvascular complications of diabetes.Nowadays,methods of therapy are limited because the pathogenesis of DN is not completely clear yet. At present research,mitogen-activated protein kinase pathway(MAPK), which is a common signal pathway in eukaryto that can be activated by numerous extracellular factors like oxidative stress,advanced glycation end products (AGEs),angiotensinⅡand lots of cytokine, should be involved in the development of DN.Extracellular signal-regulated kinasel/2(ERK1/2),the most well characterized member of the MAPKs family,is dephosphorylated mainly by MAPK phosphateases,whose expression induce downstream of ERK1/2 activation.Persistent ERK1/2 activation is accompanied by decreased Mitogen-activated protein kinase phosphatase-1(MKP-1) protein levels.Recent studies have shown that ubiquitin-proteasome pathway(UPP) is the main specific intracellular protein degradation including MKP-1 protein.However, whether UPP involves in the pathogenesis of DN via MAPK pathway or not has not been reported.To evaluate this possibility and investigate the relationship of UPP and the pathogenesis of early DN,we injected streptozotocin(STZ) to rats to induce diabetes model and used MG132,a inhibitor of UPP,to block the ubiquitination degradation of ERK1/2/MKP-1 signal pathway inhibitory protein MKP-1.In order to reveal a new theory and method to prevent and cure diabetic nephropathy,urine protein, renal staining and expressions of FN mRNA, ERK1/2 and MKP-1 we observed. Methods:1.Establishment of diabetic nephr-opathy rat model:50 merely healthy male rats of about 200 grams body were weighted and fed on standard rat food and tap water during the whole study period.One week later,they were randomly divided into two groups:normal control group(NC group,n=10) and diabetic model group(n=40).Rats in model group were established by intraperitoneal injection streptozotocin(STZ) 60 mg/kg(dissolved in pH 4.5, 0.1mmol/L citrare buffer) one by one. Meanwhile the rats in normal control group were injected the same volume of citrate buffer.72 hours after STZ injection,measuring the tail vein fasting blood glucose and the blood glucose above 16.7mmol/L were regarded as diabetes.Then the diabetic rats were randomly divided into three groups:MH group(treated with MG132, 0.1mg/kg every day,n=10),DC group(diabetic control group,n=10) and ML group(treated with MG132,0.05mg/kg every day,n=10).Meanwhile,NC group and DC group were injected with the same volum of 0.9%NS every day.2.Detection:detecting the 24-hour urinary albumin,urine protein/urine creatinine concentration and fasting blood glucose;Masson staining and electron microscopy were used to observe pathologic changes in renal tissue;Western blot was used to detect protein expression of t-ERK1/2 and p-ERK1/2 and MKP-1;RT-PCR was used to detect the expression of fibronectin(FN) mRNA.3.Statistical Analysis:Conti-nuous data was expressed as means±SEM.Multiple group comparisons were made by one-way ANOVA analysis with Student-Newman-Keuls post hoc analy-sis.Times in the same group comparison were made by Student's t test.Statistical significance was accepted at P＜0.05.Results:l.①Fasting blood glucose(FBG):compaired with NC group,FBG was higer in model group(P＜0.05);②Weight:rats in model group were loss weight(P<0.01) compared with NC group;MH group and ML group were increased weight(P＜0.05) compared with DC group;this change in MH Group was significant compared with ML group(P<0.05) also significant on 8 week compared with 6 week(P<0.05).③24-hour urinary albumin(UAER), urine protein/urine creatinine concentration(Up/Ucr):Compared with NC group, the UAER and Up/Ucr of model group increased significantly(P<0.05);while compared with DC group, they were decreased in MH group and ML group(P＜0.05);Compared with ML group, they were decreased in MH group(P＜0.05);But there were no statistical significances between on 8 week and 6 week in each group.2.Renal pathological examination:No significant changes in NC group;Tubular expanded in group DC on 6 week;no significant stromal hyperplasia.Glomerular was voluminous on 8 week.The pathological change in MH group and ML group was not obvious.3.Electron microscopy:No signifycant abnormalities of renal was indicated in NC group;in DC group,we could see the irregular thickness of the renal basement membrane,endothelial cellulan edema,widen and fusion of foot process;these changes were not obvious in group MH and ML group.4.t-ERK1/2, p-ERK1/2 and MKP-1 expression detected by Western blot:①Total ERK1/2 levels were unchanged;whereas p-ERK1/2 increased on 6 week and obviously increased on 8 week in all groups except the NC group(P<0.05); but they reduced in MH and ML group compared with DC group(P<0.05); MH group was decreased especially than ML group(P＜0.05).②Expression of MKP-1 protein was decreased in each model group compared with NC group(P＜0.05);but there was increased expression in MH group and ML group compared with DC group (P＜0.05),especially in MH group(P＜0.05).5.RT-PCR detecting FN mRNA expression:the FN mRNA expression on 6 week was increased in DC group (P＜0.01) and obviously increased on 8 week(P＜0.05) compared with NC group; but in MH group and ML group,it was reduced(P＜0.05) compared with DC group(P＜0.05);especially significant in MH group compared with ML group(P＜0.05);there was no sense of time depending.Conclusion:1.Ubiquinti-nation of MKP-1 is involved in the pathogenesis of diabetic nephropathy via up-regulation MAPK pathway.2.Proteasome inhibitor MG132 can block the degradation of MKP-1,increase the expression of MKP-1 protein,inhibit the activation of MAPK pathway and improve the early stage of DN.