Mechanism of macrophage pyroptosis induced by porcine-derived E.coli HPI via NLRP3/ASC/Caspase-1 pathway
In order to investigate the effect of porcine-derived E.coli high pathogenicity island(HPI)on pyroptosis of macrophages,endotoxin(LPS)combined with adenosine triphosphate(ATP)was used to establish a positive control of pyroptosis,and macrophages were infected with E.coli WT strain(E.coli HPI)and the previously constructed E.coli HPI knockout strain(E.coli ΔHPI).Cells were collected at different time points after infection,and cell membrane damage was determined by PI staining.RT-qPCR was used to determine the mRNA levels of key factors in the pyrosis pathway of NLRP3/ASC/Caspase-1.The assembly of inflammatory complexes of NL-RP3 and Caspase-1 was observed through confocal microscopy.Western blot was used to determine the expression of GSDMD and cleaved-GSDMD.The results showed that the mRNA levels of the key factors NLRP3,ASC,Caspase-1,GSDMD and inflammatory factors IL-18 and IL-1β of E.coli HPI+pyrosis pathway were increased compared with the E.coli ΔHPI group.The expression of NLRP3 and Caspase-1 proteins increased,and colocalization was observed under confocal microsco-py.The expression levels of GSDMD and its active form,cleaved-GSDMD,were higher in the E.coli HPI+group than in the E.coli ΔHPI group.The above results suggest that E.coli HPI in-fection of macrophages induces the assembly of NLRP3 and Caspase-1 inflammatory complexes and promotes the expression of cleaved-GSDMD by upregulating the level of key factor mRNA in the NLRP3/ASC/Caspase-1 pathway,thereby exacerbating cell membrane damage and ultimately inducing pyroptosis in macrophages.
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