| Enterotoxigenic Escherichia coli(ETEC)is a type of enteropathogenic bacteria that cause post-weaning diarrhea(PWD)in piglets,causing huge economic losses to the pig industry.In-depth exploration of ETEC pathogenic mechanism and the interaction mechanism between ETEC and host cells is crucial for better prevention and treatment of diarrhea in weaned piglets.Pyroptosis is a form of programmed cell death,which is mediated by the shearing of Gasdermin family members through inflammatory vesicles,resulting in cell membrane permeabilization,cell death and release of inflammatory factors.It has not been reported that ETEC infection could induce pyroptosis of porcine intestinal epithelial cells(EPEC-J2)yet.In this study,the inflammatory models of ETEC-infected IPEC-J2 cells was established.and the microscopic morphology of the cells was observed using transmission electron microscopy.Compared with control cells,the inflammatory model cells underwent obvious swelling,membrane perforation,rupture,and release of intracellular contents;meanwhile,qRT-PCR and Western blot results showed that the mRNA and protein expressions of NLRP3,Caspase-1 and GSDMD were significantly higher in the ETEC-infected inflammatory cells compared to the control cells,and the cleavage of Caspase-1 and GSDMD was activated.In addition,the percentage of pyroptotic cells was detected by flow cytometry,which showed that the caspase-1 signal intensity was significantly enhanced in the infection group and the percentage of pytoptotic cells cells was significantly higher than that in the control cells.In order to identify the key molecules involved in the process of ETEC-induced pyroptosis of IPEC-J2 cells,a transcriptomic analysis of the inflammatory model of ETEC-infected IPEC-J2 cells was performed by high-throughput sequencing.The reliability of the sequencing results was verified by RT-qPCR of 10 randomly selected differentially expressed mRNAs.A total of 529 differentially expressed mRNAs were identified by differential expression analysis,of which 236 were up-regulated and 293 were down-regulated;GO functional enrichment analysis revealed that the differentially expressed mRNAs were mainly enriched in cellular components,cellular processes,metabolic processes and enzyme binding reactions.The KEGG pathway enrich in mTOR signaling pathway,Toll-like receptor(TLR)signaling pathway,adhesion junctions,cell cycle and bacterial invasion of epithelial cells,demonstrating that the regulatory network of ETEC infection of IPEC-J2 cells is related to classical immune signaling pathways and structural and barrier function pathways of the small intestinal mucosa.High-throughput sequencing showed that HSP90AA1 was significantly upregulated with the inflammatory response,suggesting that HSP90AA1 may be involved in ETEC-induced pyroptosis.To prove this hypothesis,IPEC-J2 cell models with HSP90AA1 overexpression or knockdown were established by transfection of HSP90AA1 overexpression vector or siRNA,followed by ETEC infection.The morphology of HSP90AA1 knockdown cells were similar with control cells;the mRNA and protein expressions of pyroptosis related proteins NLRP3and GSDMD were significantly down-regulatedt;and the release of LDH,IL-1β and IL-18 in cell supernatants was significantly reduced;The percentage of pyroptotic cells was also significantly reduced via flow cytometry detection.In contrast,the microscopic morphology of ETEC-infected HSP90AA1 overexpression cells was severely damaged,with cell membranes rupture,disintegration of cell structure,and mitochondrial vacuolation;the mRNA and protein expressions of NLRP3 and GSDMD were significantly upregulated,and the cleaved GSDMD-N and Caspase-1 p20 proteins were sinificantly increased;the expression of LDH,IL-1β and IL-18 in cell media was significantly increased;the percentage of pyroptotic cells was significantly higher.To further verify whether the regulatory effect of HSP90AA1 on pyroptosis induced by IPEC-J2 cells depends on downstream NLRP3,NLRP3 was knock down in HSP90AA1 overexpression cell model,and the pyroptosis after ETEC infection was characterized.Compared with control cells,the pyroptotic morphology of IPEC-J2 was improved in knockdown NLRP3 group,the content of LDH,IL-1β and IL-18 in the cell supernatant,and the expression level of pyroptosis-related protein was downregulated,The above results confirmed that HSP90AA1 promoted pyroptosis induced by ETEC infection of IPEC-J2 cells through the NLRP3/Caspase-1/GSDMD classical pyroptosis pathway.In conclusion,this study has elucidated the host key regulatory molecule HSP90AA1 and related signaling pathways in ETEC-induced pyroptosis in IPEC-J2 cells,providing a theoretical basis for exploring the pathogenesis of ETEC and finding new methods to prevent and control piglets diarrhea. |
