High Glucose And Lipopolysaccharide Induces Activation NOD1-RICK-NF-κB Signaling Pathway In Cultured Rat Mesangial Cells

Objective: Diabetic nephropathy (DN) is one of the mostprevalent and serious microvascular complications in diabetes, however, itsspecific pathogenesis is still unclear. Untill now,a large number of studieshave shown that inflammation is a central mechanism of the development ofdiabetic nephropathy and the NF-kappa B signaling pathways mediateinflammation which is involved in diabetic nephropathy (DN).Recently,anew study suggests that NOD1,one of pattern recognition receptorsis involedin the NF-kappa B signaling pathway and plays an important role in thedevelopment of insulin resistance. Extracellular stimuli can transitNOD1-RICK-IκB and other signaling molecules to activate NF-κB signalingpathway. If NOD1mediated NF-κB signal pathways are involved in thepathogenesis of diabetic nephopathy is not known. In this study differentconcentrations of glucose, lipopolysaccharide (LPS)and NOD1inhibitor(ML130) were used to stimulate rat glomerular mesangial cells (HBZY-1)and the protein and gene expression of NOD1, RICK, I kappa B, NF-κBexpression were detected.The purpose of this study is to explore the role ofNOD1in NF-κB signal activation in diabetic nephropathy and find a newtheory for prevention and treatment of diabetic nephropathy. Method:1.Cultured rat mesangial cells (HBZY-1) is stitumilated with glucose, LPS and NOD1inhibitors,ML130,and divided into seven groups,as the following:(1) normal group (NC group): medium containing5.6mmol/L glucose;(2)high glucose group (HG1, HG2and HG3group):glucose concentration inmedium was10mmol/L,20mmol/L and30mmol/L,respectively;(3) osmoticgroups: medium containing5.6mmol/L glucose and24.4mmol/L mannitol.(4)LPS group (LPS1, LPS2and LPS3group): LPS concentrations in mediumwere1μg/L,5μg/L and10μg/L respectively;(5) high glucose+LPS group;medium containing30mmol/L glucose and5μg/L LPS (6) high glucose+ML130group; containing30mmol/L glucose medium previously added30μmol/L ML130(NOD1inhibitors);(7) LPS+ML130group; Containing5μg/L LPS medium previously added30μmol/L ML130;2.Immunoblotting (Western-blotting) and RT-PCR were used to detecte theprotein and gene expression of NOD1, RICK, IκB, NF-κB,3. Statisticlanalysis is used by SPSS17.0statistical software. Data were expressed asmean±standard deviation(x±s)that the comparison between groupsANOVA, pairwise comparisons using LSD t test, test level α=0.05, P <0.05was considered statistically significant. Results:1.Compared with NC group,high glucose increased the protein and mRNA expression of NOD1,RICK,NF-κB p65and decreased the expression of IκBα in mesangial cells(P<0.05) in time and concentration-dependent manners.2.Compared withNC group, LPS increased the protein and mRNA expression of NOD1,RICK,NF-κB p65,but decreased the expression of IκBα in mesangial cell (all P<0.05).3.Compared with high glucose group or LPS gourp, high glucoseand LPS synergistically increased the protein and mRNA expression ofNOD1, RICK,NF-κB p65,but decreased the expression of IκBα in mesangialcell (all P<0.05).4. Compared with high glucose group or LPS group, cellspretreated with ML130can blocke the upregulation of NOD1, RICK, NF-κBand downregulation of IκBα induced by high glucose and LPS (allP<0.05),sugesting high glucose and LPS activate the NF-κB signalingpathway via NOD1receptor.Conclusion: High glucose and LPS canactivate NOD1-RICK-IκB-NF-κB signaling pathway, via NOD1receptor andmay participate diabetic nephropathy.