The Research On Preliminary Mechanism Of Pyroptosis In Small Intestinal Epithelial Cells Induced By Enterotoxin Escherichia Coli

Background: Enterotoxin Escherichia coli(ETEC)is one of the main pathogens causing diarrhea in children under 5 years old,international travelers and newborn animals in developing countries.An estimated 840 million people worldwide are infected each year,with over 3.8 million deaths,causing significant losses to the global pig industry each year,estimated to reach tens of billions of dollars.Therefore,further research on the pathogenic mechanism of ETEC is of great significance.Research has shown that ETEC infection can cause apoptosis in host intestinal cells and induce the release of inflammatory factors.In our previous work,we found that ETEC can induce the activation of GSDMD in small intestinal epithelial cells,showing pyroptosis related phenotypic characteristics,but the specific mechanism is still unclear.Pyroptosis is a kind of programmed cell death which has been identified in recent years.Pyroptosis is often accompanied by the release of inflammatory factors,so it is highly inflammatory.Unlike apoptosis,when cells undergo pyroptosis,the permeability of the cell membrane changes,and a large amount of cell content are released,along with inflammatory factors.The mechanism exhibits a series of morphological changes and pathological characteristics related to inflammatory reactions.At present,the molecular and cellular mechanisms of pyroptosis have become one of the frontier and hot issues in the international scientific community.With the discovery of inflammasome complex and the discovery of non-canonical inflammasome pathway,pyroptosis has been redefined as a kind of programmed cell death and inflammasome dependent mechanism.Current research has revealed that the Gasdermin protein family serves as its executing molecules during cell pyroptosis and is closely related to many inflammatory diseases occurrence.Objective and Methods: To reveal the mechanism of ETEC-induced pyroptosis in small intestinal epithelial cells and provide a new target for clinical treatment.In this study,we took MODE-K cell as the experimental object.We intend to establish a model of ETEC infecting small intestinal epithelial cells,and use RNA seq,fluorescent quantitative PCR,Western blot(WB)and other methods to detect the expression of pyroptosis pathway related genes and proteins.Results:(1)The MODE-K cell model induced by ETEC was established and analyzed,the results showed that ETEC induced pyroptosis by activating GSDMD in MODE-K;(2)The results of RNA seq,q-PCR and WB showed that ETEC induced the up regulation of genes and proteins related to NLRP3,caspase-4 and GSDMD in MODE-K.Conclusion: ETEC can induce pyroptosis of MODE-K by activating GSDMD.ETEC infection affects the upregulation of NLRP3,caspase-4,and GSDMD gene expression,as well as the upregulation of NLRP3,cleavaged caspase-4(Cleaved CASP4),and cleavaged GSDMD(GSDMD-N)protein expression in MODE-K cells.We speculate that ETEC can activate GSDMD by activating caspase-4 in intestinal epithelial cells,and the activated GSDMD executes pyroptosis and causes subsequent inflammatory reaction.The results of this study further reveal the mechanism of ETEC causing intestinal diseases,and provide new theoretical basis for new or potential drug targets and related therapeutic drugs or protocols.