Astragalosides Ⅳ Improves Palmitic Acid-induced Renal Tubular Epithelial Cells Injury Due To Inhibition Of NLRP3 Inflammasome

Part 1Objective:To explore the effect and mechanism of palmitic acid(PA)on HK-2 cells of inflammation and oxidative stress at different times,and provide a theoretical basis for AS-IV prevention and treatment of diabetic nephropathy(DN).Methods:HK-2 cells were cultured in vitro and grouped as follows: Bovine serum albumin(BSA)control group;PA(6,9,12,24 h)groups.CCK-8 detected cell viability;Oil red staining observed intracellular lipid deposition;Annexin-Ⅴ-FITC/PI observed cell apoptosis;Reactive oxygen species detection kit assessed intracellular ROS expression;ELISA kit was used to detect cell proteins GSH and SOD as well as the expression of IL-1β and IL-18 in the cell supernatant;Western blot was used to determine the expressions of NLRP3,ASC,caspase-1 and NOX4 in HK-2 cells.Results:The results showed that 200 μmol/L palmitic acid(PA)can induce the viability of HK-2 cells.And with the increase of time,the cell survival rate decreased and the number of cell apoptosis increased significantly.At the same time,PA exposure significantly reduced the expression of GSH and SOD.Intracellular lipid deposition and the expression of IL-1β and IL-18 also gradually accumulated with the induction time.The expression of NLRP3,ASC,caspase-1 and NOX4 in HK-2 cells increased after PA induction.Conclusion:The mechanism of palmitic acid(PA)inducing HK-2 cells damage may be related to the oxidative stress and inflammation in the cells after PA stimulation.Thus,NOX4-mediated activation of the NLRP3 inflammasome signaling pathway may be involved in the damage of HK-2 cells induced by PA.Part 2Objective:To study the specific mechanism of AS-IV to improve HK-2 cells damage through oxidative stress and inflammation.Methods:HK-2 cells were cultured in vitro and grouped as follows: Bovine serum albumin(BSA)control group,PA group,AS-IV(10,20,40 μmol/L)administration groups,Apo(100 μmol/L)administration group.CCK-8 detected cell viability;Annexin-Ⅴ-FITC/PI observed cell apoptosis;Reactive oxygen species detection kit detected intracellular ROS deposition;ELISA kit was used to detect cell proteins GSH,SOD as well as the expression of IL-1β and IL-18 in the cell supernatant;Western blot was used to determine the expression of related proteins NLRP3,ASC,caspase-1,NOX4 in HK-2 cells.Results:AS-IV(10,20,40 μmol/L)significantly increased cell survival rate and the expression of GSH and SOD.It can not only reduce intracellular lipid deposition and apoptosis,but also significantly reduce intracellular ROS deposition and IL-1β,IL-18 expression.After administration of AS-IV and Apo,the expression of NLRP3,ASC,caspase-1 and NOX4 was significantly reduced.Conclusion:AS-IV has a protective effect on PA-induced damage to HK-2 cells.The protective mechanism of AS-IV may be due to the ability of AS-IV to reduce oxidative stress and inflammation.That is,AS-IV may play a key role through NLRP3 complex pathway induced by NOX4 and provide new ideas for the treatment of kidney disease.Part 3Objective:To study the mechanism of oxidative stress and inflammation in renal tubular injury with T2 DM rats,and to further explore the effect of AS-IV on it in the process of renal injury.Methods:The experimental rats were divided into 4 groups: Normal group,Model group,AS-IV group(80 mg/kg)and Metformin group(200 mg/kg).After the experiment,the kidneys were collected.And HE staining was used to observe the morphological changes of the renal tubules in each group;Oil red staining was used to detect lipid deposition;Immunohistochemistry(IHC)was used to evaluate the NOX4 and NLRP3 protein in the renal tubules;Western blot was used to assess the expression of NLRP3,ASC,caspase-1,NOX4,IL-1β,p22 phox and p47 phox.Results:Oil red staining showed that compared with the normal group rats,the model group had obvious orange-red lipid droplets.After AS-IV administration,the lipid deposition in the renal tubules was significantly reduced;HE staining displayed that the model rats showed severe pathological changes,such as vacuolar degeneration and damaged lumen.After the administration,the renal tubular lesions were significantly improved;The IHC results showed that compared with the normal group rats,the expressions of NOX4 and NLRP3 proteins in the model group increased significantly.After administration of AS-IV,the expressions of NOX4 and NLRP3 decreased.WB results showed that compared with the normal group,the expressions of NLRP3,ASC,caspase-1,NOX4,IL-1β,p22 phox and p47 phox proteins in the model group increased significantly;The expressions of these related proteins were reduced after AS-IV(80 mg/kg)treatment.Conclusion:AS-IV can improve renal pathological changes,and reduce lipid deposition Beside that it also can reduce the expressions of NLRP3,ASC,caspase-1,NOX4,IL-1β,p22 phox and p47 phox indicating that AS-IV may inhibit NOX4-mediated NLRP3 inflammasome signaling pathway and plays a protective role.