Role Of NOX4 In Regulating NLRP3 Inflammasome Expression In High Glucose-induced Podocyte Apoptosis

Background and objective:Diabetes mellitus(DM)is a common chronic disease characterized by disorders of blood glucose metabolism.As a serious microvascular complication of diabetes,diabetic nephropathy(DN)has become the first cause of end-stage renal disease in western countries and first-tier cities in China.Hyperglycemia,as the core link of DN,can activate oxidative stress and inflammatory response to induce podocyte apoptosis,but the specific mechanism remains unclear.The concept of inflammatory corpuscles was first proposed by the Tschopp research team in 2002.They are composed of nucleotide-binding oligomerization domain-like receptor family members NLRP3,adaptor-apoptosis-associated speck-like proteins(ASC)and effector protein(caspase-1),involved in the development of type 2 diabetes,autoinflammatory reactions,atherosclerosis and other diseases.Under normal circumstances,NLRP3 inflammasome is at rest,and various endogenous substances such as adenosine triphosphate,uric acid crystals,reactive oxygen species,?-amyloid and lysosomal enzymes,and exogenous substances alum,asbestos and silica activate the NLRP3 inflammasome.NLRP3 rapidly binds to ASC to activate caspase-1,which is involved in the regulation of the secretion of pro-inflammatory factors such as IL-1?,IL-18 and IL-33.NLRP3 inflammasome is expressed in renal intrinsic cells such as glomerular endothelial cells,renal tubular epithelial cells and podocytes,and participates in diabetic nephropathy,focal segmental glomerulosclerosis,IgA nephropathy,acute kidney injury and acute pyelonephritis.And the development of a variety of kidney diseases.Oxidative stress is a pathological state in which the active redox(mainly ROS-based)is produced by the imbalance of redox reaction in the body,and participates in the onset and development of diabetic nephropathy.ROS are mainly derived from mitochondria,and can also be derived from reduced nicotinamide adenine dinucleotide phosphate oxidase(NADPH),xanthine oxidoreductase,lipoxygenase and cyclooxygenase.Among them,NADPH oxidase is mainly expressed in the kidney.It is the main source of ROS production in the renal cortex.The activation pathways of NLRP3 inflammasome mainly include: 1)intracellular potassium efflux;2)lysosomal pathway;3)ROS production.Among them,ROS gradually became a common pathway for NLRP3 inflammasome activation.Studies have shown that hyperhomocysteinemia stimulates NADPH oxidase-derived large amounts of ROS to trigger the formation and activation of NLRP3 inflammasome,resulting in IL-1? and other factors that cause podocyte damage.However,it has not been reported whether NOX4-mediated NLRP3 inflammasome activation is involved the apoptosis of HPC in Hyperglycemia.In this study,human glomerular podocytes were used as the research object.The effects of high glucose and NOX4 specific inhibitor GKT137831 on apoptosis of human glomerular podocytes(HPC)were observed in high glucose environment in vitro.The NLRP3 inflammasome activates a possible mechanism involved in apoptosis of podocytes.Therefore,the mechanism of early diabetic nephropathy and new therapeutic targets for delaying the progression of diabetic nephropathy are explored.Methods:HPC was routinely cultured in vitro,and the experimental groups were: 1,normal sugar group;2,mannitol group;3,high glucose group;4,high glucose + inhibition group.The concentration of glucose and inhibitor was set according to the pre-experimental results.At the end of the experiment,the expression of ROS in podocytes was detected by fluorescence microscopy.The apoptosis rate of podocytes was detected by flow cytometry.Western-Blot assay was used to detect the expression of NOX4 protein,NLRP3 inflammasome protein(NLRP3,ASC,caspase-1),podocyte-specific protein(nephrin,podocalyxin)and apoptosis-related proteins(Bcl-1,Bax).Results:Compared with the normal sugar group,high glucose increased the expression of NOX4 and NLRP3 in the podocytes,and the difference was statistically significant(P<0.05).Compared with the normal sugar group,the expression of ROS in the podocytes of the high glucose group increased,the expression of the inflammasomeassociated protein(NLRP3,ASC,caspase-1)and the apoptotic protein Bax increased,while the podocyte structural proteins(nephrin,podocalyxin)and the expression of anti-apoptotic protein Bcl-1 is decreased,and the apoptosis rate of podocytes is increased.Pretreatment of GKT 137831 can attenuate ROS expression in podocytes,reduce the expression of inflammation-related proteins(NLRP3,ASC,caspase-1)and apoptotic protein Bax.And partially restored the expression of podocyte structural protein(nephrin,podocalyxin)and anti-apoptotic protein Bcl-1,and decreased the apoptosis rate of podocytes.Conclusions:1.High glucose induces high expression of NOX4 and activation of NLRP3 in human glomerular podocytes.2.Inhibition of NOX4 activity can reduce oxidative stress and inflammatory response in podocytes induced by high glucose,and reduce the apoptosis of podocytes induced by high glucose.