The Regulating Effect Of CYLD On Activation Of NF-?B Inflammatory Signaling In Mesangial Cells Induced By High Glucose

Objective: Diabetic nephroapathy is an severe microvascular complication of diabetes.In the west,it is the main cuase lead to end stage renal disease(ESRD)and ultimately depend on dialysis to sustain life.A number of studies revealed that the mononuclear macrophage and increased inflammatory cytokines can be seen in early diabetic renal tissues.Nuclear factor ?B(NF-?B),the key part of multiple signal transduction of inflammation,play a crucial role in mediating many inflammatory response.Our previous study has found ubiquitin modification take part in activation of NF-?B signaling pathway.However,deubiquitin modification also involve in regulation of signaling pathway and keep dynamic balance with it.Cylindromatosis(CYLD)is one of the main negative regulatory factors and inflammation inhibiting factor in NF-?B signaling pathway.What's the function of CYLD in diabetes is rarely reported.In this study,we observed the espression of CYLD ? total I?B ? ? phosphorylation NF-?B p65 ?phosphorylation I?B??IKK??MCP-1?IL-6?IL-8 in cultured mouse mesangial cells stimulated by high glucose at first,and then used the techniques of SiRNA interference and overexpression transfection of CYLD to explore the role of CYLD in diabetic nephroapathy.Methods:Cultured mouse GMCs(SV40)were devided into six groups:1?normal group(contains 5.6mmol/L glucose);2?Different concentration of glucose groups(contains 10?20?30 mmol/L glucose in each group);3?Osmotic pressure group(contains 5.6mmol/L glucose and 24.4 mmol/L mannitol);4 ? CYLD SiRNA interference group: 100nmol/L CYLD SiRNA interference sequence added in medium;5 ? CYLD transfection group: 1*108 TU/ml CYLD lentivirus added in cultured medium,the multiplicity of infection of mouse GMCs is 50;6?blank load transfection group:virus CON235 without CYLD was served as negative control added in medium use the same concentration.Each group was induced 6?12?24?48?72 hours by high glucose,the expression of CYLD? I?B??p-NF-?B p65?p-I?B??IKK??MCP-1?IL-6?IL-8 were detected by Western Blot?RT PCR and ELISA.Result:1?The expression of CYLD?total I?B? were decreased and p-NF-?B p65?p-I?B??MCP-1?IL-6?IL-8 were inhanced by high glucose in a time and dose-dependent manner(p<0.05);2?High glucose had no obvious effect to the expression of IKK? protein and mRNA(p>0.05);3?The expression of p-NF-?B p65?p-I?B??MCP-1?IL-6?IL-8 were significantly increased but total IKB?challenge with CYLD Si RNA sequence compared with normal group(p<0.05);4 ? On the contrary,CYLD transfection inhanced the espression of total I?B? and reduced the expression of p-I?B??p-NF-?B p65?MCP-1?IL-6?IL-8(p<0.05).Conclusion: 1?high glucose can activate the downstream inflammatory factors of NF-?B signal pathway such as MCP-1?IL-6?IL-8;2?CYLD can negatively regulate activation of NF-?B pathway induced by high glucose;3 ? High glucose could activate NF-?B pathway by downregulation of CYLD,and downregulation of CYLD maybe play a role in pathogenesis of diabetic nephropathy.