The Protective Mechanism Of Gushen Xiezhuo Heluo Prescription Against Podocyte Injury Induced By High Glucose By Inhibiting The Activation Of NLRP3 Inflammasome

Diabetic Kidney Disease comes to be the main cause of end-stage renal disease in our country.Podocyte injury is a key factor in the generation of proteinuria and glomerular sclerosis in diabetic nephropathy.NLRP3 inflammasome,as a key molecular junction in the regulation of chronic microinflammation,is directly involved in the pathological process of the injury of podocytes in DKD.Objective:1.To investigate the mechanism of activation of NLRP3 inflammasome,expression of Nephrin and Podocin protein and apoptosis in murine podoc5 cells;2.To study the expression of reactive oxygen species in MPC5 cells,the expression of NLRP3 inflammasome related molecular signals,the expression of Nephrin and Podocin proteins and the apoptosis of MPC5 cells stimulated by High Glucose(HG);3.To study the protective effects of GSP on the activation of NLRP3 inflammasome in MPC5 cells and podocyte injury by HG.Methods:Mouse podocytes(MPC5 cells)were cultured in vitro,and intervention was performed according to the following groups:Control Group,without glucose and test materials;NG Group,5mM Glucose is added;Mannitol control group,5mmol/L glucose and 24.5mmol/L Mannitol;HG group,30mM Glucose.In the high glucose+Middle dose of GSP(M-GSP)group,30mmol/L glucose+1 mg/mL GSP extract were added;In the High glucose+High dose of GSP(H-GSP)group,30mmol/L glucose+2 mg/mL GSP extract were added.CCK8 assay was used to determine podocyte activity.The migration ability of podocytes was determined by cell scratch assay.5.ROS levels of podocytes were detected by DCFH-DA fluorescent probe.Cell apoptosis was detected by flow cytometry with Annexin V-EGFP fluorescence staining.The expression of IL-1β and IL-18 in podocytes was detected by ELISA.The Nephrin,Podocin and NLRP3,ASC and Caspase-1 expressions were analysed by WB Analysis.Results:30mmol/L glucose treatment for 72 hours could induce apoptosis of podocytes and increase the expression of intracellular reactive oxygen species.The protein expressions of Nephrin and Podocin in HG group were decreased,while the expressions of NLRP3,ASC,caspase-1 and IL-1β in HG group were increased,and the differences were statistically significant compared with NG group(*P<0.05,**P<0.01).The apoptosis in M-GSP and H-GSP groups was reduced,the expression of reactive oxygen species was decreased,the protein expression of Nephrin and Podocin in podocytes were increased,and the high expression of NLRP3,ASC,Caspase-1 and IL-1β were inhibited.No expression of IL-18 was detected in all groups.Conclusion:1.High glucose stimulated MPC5 cells could increase intracellular reactive oxygen species and activate NLRP3 inflammasome,which was manifested by increased protein expressions of NLRP3,ASC,caspase-1 and IL-1β decreased protein expressions of Nephrine and Podocin,and increased cell apoptosis.2.GSP can inhibit podocyte injury induced by high glucose.Its mechanism may be related to inhibiting the production of reactive oxygen species,inhibiting the activation of NLRP3 inflammasome,reducing the expressions of NLRP3,ASC,caspase-1 and IL-1 β,and increasing the protein expressions of Nephrin and Podocin.3.GSP may improve the podocyte injury,which may be by inhibiting the ROS level and the activation of NLRP3 inflammasome.