Experimental study on the expression of focal death-related proteins in in-vitro oxidative stress models and the protective effect of disulfiram

CHENG Hualei; WANG Yajun; XING Weiwei

doi:10.3969/j.issn.1672-2922.2026.01.012

Abstract：Objective To investigate the expression regulation mechanism of pyroptosis-related proteins in an oxidative stress model of H2O2 induced HEI-OC1 cells,and the effects and mechanisms of disulfiram on pyroptosis-related signaling pathways.Methods HEI-OC1 cells were treated with H2O2 disulfiram or H2O2 combined with disulfiram,and cell viability was detected by cell counting kit-8.ROS generation in cells was detected using flow cytometry and immunofluorescence techniques.The expression of key proteins NLRP3,caspase-1,and N-GSDMD pyroptosis was detected by immunofluorescence.and mRNA expression levels of pyroptosis-related genes NLRP3,caspase-1,GSDMD,IL-1β,and IL-18 by RT-qPCR.Morphological changes of cells were examined by scanning electron microscopy.Levels of inflammatory factors IL-1β and IL-18 in cell culture supernatant were detected by ELISA.Results HEI-OC1 cells inhibitory concentration(IC50)after 4 h of H2O2 exposure was 829.5 μmol/L,and cell viability was observed at 5 μmol/L disulfiram,so 5 μmol/L disulfiram+800 μmol/L H2O2,ROS were significantly increased after treatment with H2O2(P<0.001),together with NLRP3 caspase-1,N-GSDMD protein expression(P<0.0001)and elevated levels of factors IL-1β,IL-18 in the cell culture supernatant(P<0.001),as well as mRNA expression levels of LRP3,caspase-1,GSDMD,IL-1β and IL-18(P<0.001).Scanning electron microscopy showed swollen cells with holes and a large number of vesicular protuberances.When treated with H2O2 combined with disulfiram,ROS generation was significantly lower(P<0.01),with significantly less expression of pyroptosis-related proteins(P<0.05),less mRNA expression of NLRP3,caspase-1,GSDMD,IL-1β,IL-18 were decreased(P<0.05),and less elevation of inflammatory factors IL-1β,IL-18 in the culture supernatant(P<0.01),with scanning electron microscopy showing less holes cells and less vesicular protuberances on cell membrane.Conclusions H2O2 can induce oxidative stress response and trigger cell pyroptosis in HEI-OC1 cells,and disulfiram can reduce H2O2-induced HEI-OC1 cell injury by inhibiting the generation of ROS and activation of NLRP3 inflammasome pathway.