Cochlear Marginal Cells Pyroptosis Is Induced By Cisplatin Via NLRP3 Inflammasome Activation

Objective: To investigate the role of NLRP3-mediated inflammatory response in marginal cells(MCs)pyroptosis induced by cisplatin and the regulation of TXNIP.Methods:(1)The primary MCs of rat cochlea were treated with a gradient concentration of cisplatin for 24 h,48h,and 72 h,respectively.The survival rates of cells of each group were detected by the cck-8 method and flow cytometry.(2)Primary MCs were divided into the control group,cisplatin group,NLRP3 transfection group,and TXNIP transfection group.NLRP3 transfection group and TXNIP transfection group were down-regulated by small interfering RNA transfection method respectively and then treated with cisplatin.(3)The ultrastructural changes of cells between groups were observed under electron microscopy,and the proportions of MCs with cell membrane rupture or DNA rupture were detected by TUNEL staining and flow cytometry.The levels of m RNA and protein of NLRP3,ASC,cleaved-caspase1,IL-1β,and N-GSDMD were detected by PCR and western blot,respectively.The concentrations of IL-1β in cell supernatants were detected by ELISA.Results:(1)The treatment of 5μM cisplatin for 24 h could establish a stable model of MCs damaged by cisplatin in vitro.(2)It could be observed that the cell membrane of the cisplatin group formed pores and lost integrity under the electron microscope.but there was no such morphological change in the control group,and the change disappeared in the NLRP3 transfection group and TXNIP transfection group.(3)Compared with the control group,the number of TUNEL positive cells in the cisplatin group was significantly increased,while the number of TUNEL positive cells in the NLRP3 transfection group and TXNIP transfection group were decreased.(4)Flow cytometry results showed that the proportion of cell membrane ruptured cells(Q1+Q2)was 2.3% in the control group,8.2%in the cisplatin group,3.1% in the NLRP3 transfection group,and 4.6% in the TXNIP transfection group on average.(5)Compared with the control group,the expressions of NLRP3,ASC,Caspase-1,IL-1β,and GSDMD were significantly increased in the cisplatin group,and the expression of these indicators was also lower in the NLRP3 transfection group and TXNIP transfection group than in the cisplatin group.(6)The concentrations of IL-1β in the supernatant of control group,cisplatin group,NLRP3 transfection group,and TXNIP transfection group were 4.69±1.62pg/ml,38.08±7.15pg/ml,8.04±6.17pg/ml,and6.78±3.37pg/ml,respectively.The above differences were statistically significant(P<0.05).Conclusions: Pyroptosis is one of the MCs death modes induced by cisplatin.Activation of NLRP3 may be one of the reasons why cisplatin leads to MCs pyroptosis.The above effects of NLRP3 in cisplatin-induced MCs may be induced by activation of ASC/caspase-1/IL-1β signal.And TXNIP could regulate NLRP3 related signaling pathways.