| Objective:Diabetic nephropathy(DN)is a common and serious microvascular complication of diabetes mellitus.Recent studies have shown that Hyperglycemia,hemodynamic change is a key link in the development of DN.A large number of NF-?B and mononuclear macrophages are recruited in renal tissue,which illustrates inflammation is the a chief mechanism in the occurrence of DN.Nuclear factor kappaB(NF-?B)comprises a family of transcription factors and plays critical roles in regulating immunity and cell survival and expression of inflammatory cytokines.Actually,it has been proved that different posttranslational modifications aid in activating or inhibiting NF-?B induced by diverse agents,such as phosphorylation,ubiquitination and sumoylation.Sumoylation is the process of posttranslational convalent modification by a relatively small polypepetides called SUMO,which controls diverse cellular processes,including strengthening the protein stability,DNA repair,nucleo-cytoplasmic trafficking,signal reansduction,transcriptional regulation.SUMO attachment to substrate protein requires ATP and involves three enzymes: a heterodimeric SUMO-acticating enzyme(E1),a SUMO-conjugating enzyme(E2),and one of several SUMO ligases(E3).As an SUMO E3 ligase,the latest research proved that NF-?B activation could be enhanced by PIASy mediating NEMO sumoylation while genotoxic agents stimulating in macrophages.However,the roles of PIASy in regulating NF-kB signaling on the diabetic nephropathy and the mechanism are unclear.We detected the change of NF-?B pathway related signaling molecules(I?B??p-I?B??p-NF-? Bp65 ? IKK?)and downstream inflammatory factors(IL-6 ?MCP-1)when the rat glomerular mesangial cells were transfected PIASy-siRNA or control-si RNA,aiming to explore the mechanism of PIASy mediating the NF-kB signaling in diabetic nephropathy and find a new therapeutic targets.Methods:(1)Rat mesangial cells were divided into 5 groups: normal glucose group(5.6mmol/L),high glucose group(10,20,30mmol/L),osmotic control group.(2)The protein expression of PIASy?I?B??p-I?B??p-NF-?B ?IKK? was measured by Western-blot,while the levels of IL-6?MCP-1 in celture supernate was measured by ELISA.(3)After the cells were transfected PIASy-siRNA,rat mesangial cells were divided into 4 groups again:(1)siNC:transfected control-siRNA with 5.6mmol/L glucose;(2)siPIASy: transfected PIASy-siRNA with 5.6mmol/L glucose;(3)HG+si NC:transfected control-siRNA with 30mmol/L glucose culturing 24hours;(4)HG+siPIASy: transfected PIASy-siRNA with 30mmol/L glucose for 24 hours.The expression of PIASy?I?B??p-I?B??p-NF-?B ?IL-6?MCP-1 was measured again.The expression and location of PIASy ? IKK? were observed by immumofluorescence colocalization.Results:1.Compared with normal control group,the expression of PIASy gene and protein was enhanced after high glucose treatment,in a doseand time-dependent manner(P < 0.05).2.the expression of I?B? was decreased,while the expression of p-I?B? and p-NF-? B was up-regulated induced by different time and different concentrations of high glucose(P < 0.05).3.the expression of IKK? had no significant differences when challenging of different time and different concentrations(P>0.05).Moerover,the colocalization of PIASy?IKK?was enhanced while high glucose stimulating.4.Levels of IL-6 and MCP-1 were enhanced in high glucose group in a dose and time-dependent manner(P < 0.05).5.the expression of I?B? was up-regulated,while the expression of p-I?B? and p-NF-? B was down-regulated after the cells transfected PIASy-siRNA(P<0.05).6.the levels of IL-6 ? MCP-1 were decreased after the cells transfected PIASy-siRNA(P < 0.05).Conclusion: 1.high glucose can increase the expression of PIASy 2.High glucose decrease expression of I?B?,while induce NF-?B activation and enhance the expression of the downstream inflammatory factors.3.Up-regulation of PIASy may play a role in pathogenesis of inflammation in diabetic nephropathy by activation of NF-?B pathway. |
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