Zearalenone Induces NLRP3-dependent Pyroptosis Via Activation Of Nf-?B Modulated By Autophagy In INS-1 Cells

Objective: Zearalenone(ZEA),also known as F-2 toxin which is frequently detected in feed and food,the toxin-producing bacteria is Fusarium strains.It has been reported that mycotoxins are closely related to the development of diabetes.Moreover,it has been found that ZEA could stimulate inflammatory response,elevate autophagy level and cause cell damage.And islet inflammation,islet cell damage and autophagy are closely related to the development of diabetes.In this study,we explored the inflammatory damage and even pyroptosis caused by ZEA in INS-1 cells,analyzed the mechanism of focal growth,and explored the occurrence of autophagy and its effect on cell death.Method: INS-1 cells were chosen to be the research objects.MTT and lactate dehydrogenase release assays were used to detect the toxic effects of ZEA in INS-1 cells and the effect on survival rate.Western blot was used to detect the activation of NLRP3 inflammasomes in INS-1 cells and itsdownstream.Changes in protein expression;real-time quantitative PCR was used to detect changes in the expression levels of inflammatory genes Il-1? and TNF-? in INS-1 cells after ZEA exposure and after exposure to ZEA.Detection of intracellular pyroptosis occurred using PI/Hoechst,lactate dehydrogenase release and Western blot(caspase-1 expression)methods.The number of autophagic vesicles in the cells was detected by transmission electron microscopy(TEM),and the expression of autophagy-related proteins was detected by Western blot.Immunofluorescence and Western blot were used to detect NF-?B p65 positional translocation and activation.Lactate dehydrogenase release was used to detect cell death and content release after NF-?B inhibition.Real-time quantitative PCR was used to detect the expression of inflammatory genes Il-1? and TNF-? in INS-1 cells after NF-?B inhibition.Changes in the expression levels of inflammatory genes Il-1? and TNF-? in INS-1 cells after exposure to ZEA intervention require the pretreatment of cells and the application of ZEA stimulation.Results: Compared with the control group,the survival rate of INS-1 cells decreased after exposure to ZEA,which showed a decrease in survival rate and an increase in LDH release in MTT.ZEA induces the activation of NLRP3 inflammasome-dependent pyroptosis,which is characterized by activation of caspase-1 p20,elevation of Il-1? and TNF-? genes and protein levels,PI/Hoechst staining and LDH release,via the decline in siNLRP3 and MCC950 after treatment.ZEA induced up-regulation of intracellular autophagy,which was manifested by an increase in the number of autophagic vesicles,an increase in autophagy-related protein expression,and an autophagic flux assay by chloroquine.Pretreatment with Rapa increased the number of AVs in ZEA-treated INS-1 cells,whereas,opposite effects were displayed in 3MA group.Moreover,the result evidenced that pretreatment with Rapa down-regulated the expression of caspase-1 p20,IL-1? p17 and TNF-?,and blocked the release of LDH in ZEA-treated cells;Opposite effects in these proteins expression were displayed in 3MA group,but LDH releasewas not significantly up-regulated by 3MA.Activation and nuclear translocation of NF-?B p65 in cells can be caused by ZEA stimulation,which is manifested by increased expression of total protein p-NF-?B p65,decreased cytoplasmic NF-?B p65 and increased intranuclear.ZEA-induced NF-?B P65 level in the nuclear fraction was reduced by PDTC pretreatment and the activation of NLRP3 and pyroptosis were also reduced.Moreover,ZEA-induced nuclear translocation and activation of NF-?B p65 can be regulated by autophagy,which is shown by changes in nuclear translocation and activation of NF-?B p65 after 3MA and Rapamycin intervention.Conclusion: ZEA triggered cell death in INS-1 cells.ZEA-induced pyroptotic cell death in INS-1 cells is dependent on the activation of NLRP3 inflammasome.Exposure of ZEA elevated autophagy level in INS-1 cells and autophagy plays a protective role in ZEA-induced activation of NLRP3 inflammasomes.ZEA triggered NF-?B p65 activation and nuclear translocation,which was involved in NLRP3 inflammasome activation and pyroptotic cell death and was mediated by autophagy.