RIPK2 Mediated Autophagy And Regulate ROS-NLRP3 Inflammasome Signaling In High Glucose-induced Glomerular Mesangial Cells

Objective: Diabetic nephropathy(DN)is a major and serious microvascular complication of diabetes mellitus(DM),while the pathogenesis of DN is complexing,many factors were involved in its occurrence and development,recent studies showed that oxidative stress and inflammation may be involved in diabetic renal damage in different pathological process and play a central role,our previous study has proved that high glucose induced oxidative stress,activated reactive oxygen species(ROS)and NLRP3 inflammasome signaling pathway,results in the activation of Caspase1 and promotes the mature and secretion of proinflammatory cytokines IL-1β.Autophagy is a degradation process involved in the clearance of damaged proteins and organelles to maintain cellular homeostasis in the cell,to achieve energy recovery through the clearance of damaged mitochondria and decrease production of ROS,autophagy may be involved in the regulation of ROS-NLRP3 inflammasome signaling pathway to avoid excessive inflammation,but so far,the research of autophagy on glomerular mesangial cell(GMC)induced by high glucose and the possible regulation mechanism is rare,and the results are still controversial,some studies suggested that enhanced autophagy may reduce oxidative stress and inflammation,while excessive autophagy may also lead to autophagic cell damage and increased apoptosis.Among the limited regulation mechanism of autophagy,receptorinteracting protein 2 kinase(RIPK2),an important connexin of Nod1/Nod2 which belongs to Nod-like receptor(NLR)family,were proved to activating of NF-κB and MAPK mediated inflammation,also involved in the autophagy mediated by Nod1/Nod2,suggesting a potential role of RIPK2 in autophagy and the ROS-NLRP3 inflammasome signaling pathway,while,rare studies on exploring the relationship between RIPK2 and autophagy and the influence on ROS-NLRP3 inflammasome signaling pathway in GMCs induced by high glucose,our study first observed the expression of RIPK2,LC3II/I,ROS,Caspase1,IL-1 β when the GMCs were cultured in different time and concentration glucose,and then used the techniques of si RNA interference of RIPK2,detected the change of autophagy and key factor of ROS-NLRP3 inflammasome again,aimed to explore the potential role and mediated mechanisms of RIPK2 in autophagy and their relationship with ROS-NLRP3 inflammasome signaling of high glucose induced mouse mesangial cells,to provide new idea for prevention and treatment of DN.Methods :(1)Cultured mouse mesangial cells(SV40)were divided into the three groups: normal control group(NC group): 5.6 mmol/L glucose;osmotic control group(OP group): 5.6 mmol/L glucose +24.4 mmol/L mannitol;high glucose group(HG group): 10 mmol/L glucose(HG1 group),20 mmol/L glucose(HG2 group),30 mmol/L glucose(HG3 group);(2)The cells were cultured 0h,2h,6h,12 h,24h,48 h,72h,the protein expression of RIPK2,LC3II/I,Caspase1 and IL-1β were detected by Western-blot,mRNA expression of RIPK2,Caspase1 and IL-1β were detected by RT-PCR,autophagysome was measured using GFP-RFP-LC3 and ROS were detected by DCFH-DA,while the level of IL-1β in culture supernatant was measured by ELISA.(3)After the cells were transfected with RIPK2 siRNA,the SV40 were divided into four groups:(1)siNC group: transfected control-siRNA with 5.6 mmol/L glucose culturing 12 hours;(2)siRIPK2 group: transfected RIPK2-siRNA with 5.6 mmol/L glucose culturing 12 hours;(3)HG+siNC group: transfected control-siRNA with 30 mmol/L glucose culturing 12 hours;(4)HG+si RIPK2 group:transfected RIPK2-si RNA with 30 mmol/L glucose culturing 12 hours.The expression of RIPK2,LC3II/I,Caspase1,IL-1β,ROS and autophagysome was measured again.(4)Statistical analysis: we used statistical software SPSS 24.0 for data analysis,the data is represented by the mean and standard deviation(?),the variance among groups were analyzed by one-way ANOVA and LSD-t were used to multiple comparisons between groups,homogeneity test of variance was compared in every group,P<0.05 was considered to be statistically significant.Results:(1)The expression of ROS,caspase1 and il-1β increased with a time and dose-dependent manner in HG group compared with NC group(P< 0.05).(2)High glucose induced the up-regulation of RIPK2 and LC3 in GMCs in short term(P< 0.05)compared with NC group and reached a peak in 12 hours,whereas it decreased significantly when cultured more than 12 hours(P< 0.05)with a glucose concentration dependent manner.(3)RIPK2-siRNA transfection successfully inhibited the expression of RIPK2,decreased the expression of LC3II/I and enhanced the expression of ROS,Caspase1 and IL-1β compared with si NC group(P< 0.05).Conclusion:(1)High glucose could induce the activation of ROS-NLRP3 inflammasome signaling in mouse mesangial cells.(2)High glucose may play a dual role in RIPK2 and autophagy in GMCs,that activate RIPK2 and autophagy in short time and inhibit in long term.(3)RIPK2 negative regulates the ROS-NLRP3 inflammasome signaling through autophagy and may involve in the pathogenesis of diabetic nephropathy.