Effect of Cyclocarioside H on the Damage of Renal Podocytes in Mice Stimulated by High Glucose
[Objective]This experiment was to investigate the effect of cyclocarioside H on the damage of renal podiocytes(MPC-5)induced by high glucose by inhibiting the NLRP3/ASC/Caspase1 pathway.[Method]MPC-5 cells were incubated with different concentrations(0,3.25,7.5,15,30,60 and 120 μmol/L)of cyclocarioside H,and the cell viability was detected by CCK-8 method to determine the safe concentration.MPC-5 cells were divided into blank control(CON),D(+)-anhydrous glucose isoosmotic control(MA),high glucose(HG)and cyclocarioside H with different concentrations(3.25,7.5 and 15 μmol/L)groups(CY3.25,CY7.5 and CY15).HG and CY3.25,CY7.5 and CY15 groups were added with 30 μmol/L D(+)-anhydrous glucose,MA group was added with 5.5 mmol/L D(+)-anhydrous glucose+24.5 mmol/L mannitol,CON group was added with DMEM medium without FBS.After incubating the cells for 24 h,the culture medium of CY3.25,CY7.5 and CY15 groups was changed to different concentrations of cyclocarioside H,and the cells were incubated again for 24 h.The cells were collected,and CCK-8 and lactate hydrogenase(LDH)assay were used to determine cell viability and damage degree,respectively.The apoptosis rate was detected by fluorescent staining with propyl iodide(PI).The co-localization of NOD-like receptor heat protein domain associated protein 3(NLRP3)and apoptosis-associated speck-like proteins(ASC)in MPC-5 cells was observed by immunofluorescence.The contents of Pro-IL-1β and IL-1β in the supernatant were detected by ELISA.Western blotting was used to detect the expression of NLRP3,ASC,Caspase1/Pro-Caspase1,Desmin,Nephrin,MFF and Collagen Ⅳ proteins.[Result]Compared with 0 μmol/L cyclocarioside H group,the cell viability of 7.5 and 15 μmol/L cyclocarioside H groups was significantly increased(P＜0.05),and that of 30,60 and 120 μmol/L cyclocarioside H groups was significantly decreased(P＜0.05).Therefore,3.25-15 μmol/L was subsequently selected as the safe concentration of cyclocarioside H.The cells were treated with the above safe concentration of cyclocarioside H,and the results showed that compared with HG group,cell viability in CY3.25,CY7.5 and CY15 groups was significantly increased(P＜0.05),while LDH activity in cell supernatant was significantly decreased(P＜0.05),and in a dose-dependent manner.The apoptosis rate of CY7.5 and CY15 groups was significantly decreased(P＜0.05).The results of immunofluorescence assay showed that compared with CON group,the fluorescence of NLRP3 and ASC in HG group was enhanced and the red and green fluorescence labels overlapped.Compared with HG group,the fluorescence of NLRP3 and ASC in CY3.25 and CY7.5 groups was weakened and there was no overlap of red and green fluorescence labels,while there was no positive fluorescence of NLRP3 and ASC in CY15 group.ELISA results showed that compared with HG group,the ratio of IL-1β and Pro-IL-1β in CY15 group was significantly decreased(P＜0.05).Western blotting results showed that compared with HG group,the expressions of NLRP3,ASC,Desmin,MFF,Collagen Ⅳ and Caspase1/Pro-Caspase1 in CY15 group were significantly decreased(P＜0.05),while the expression of Nephrin protein was significantly increased(P＜0.05).[Conclusion]Cyclocarioside H could improve the damage of MPC-5 cells induced by high glucose,mainly by altering the expression of related proteins in the NLRP3/ASC/Caspase1 pathway,thereby exerting a protective effect on cells.
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