Effect Of Quercetin On Injury Of IPEC-J2 Cells Induced By Enterotoxigenic Escherichia Coli

Enterotoxigenic Escherichia coli is one of the main pathogens caused diarrhea in piglets.Adhesion plays an important role in the process of pathogenicity,which can also promote the formation of biofilms.Clinically using antibiotics leads to bacterial resistance and antibiotic residues,so it is critical to find drugs to replace antibiotics.Traditional Chinese medicine and its active ingredients have good effect on antibacterial virulence factors,without causing pressure on bacterial growth,but by regulating virulence factors,quorum sensing and biofilm formation to reduce the pathogenicity of bacteria,greatly reducing the probability of bacterial drug resistance.In this study,quercetin was selected as the research object,and the concentration of the drug that did not inhibit bacterial growth was selected to study the effect of quercetin on the transcriptome,quorum sensing,biofilm formation and inflammatory injury,which elucidated the quercetin inhibited the quorum sensing and the mechanism of inflammatory injury.The specific content of test and results are as follows:Firstly,the anti-bacterial effect of quercetin on ETEC-O139 was evaluated,and the results showed that quercetin did not significantly inhibit the growth of ETECO139 in the concentration range of 0-40 μg/m L.By using RNA-seq for sequencing,a kind of modern molecular sequencing technology,the results showed that after ETECO139 was treated with 10 μg/m L and 20 μg/m L quercetin,there were 12 and 20 differentially expressed genes respectively.The differential genes were analyzed by Gene Ontology,and it was found that quercetin in the cellular component of cell,cell part,membrane and membrane part,and macromolecular complex exerted the molecular function of binding,catalytic activity and nucleic acid binding transcription factor activity,which caused the phenotype of cellular processes,metabolic processes,responses to stimulus,biological regulation,regulation of biological process,localization,locomotion,single-organism process and multi-organism process,etc.changing in ETEC-O139.Then,the change of differentially expressed genes in pathway were analyzed.Through the KEGG public pathway database,it was found that there were differentially expressed genes in the pathways related to the bacterial chemotaxis,lipopolysaccharide biosynthesis,ABC transporters,quorum sensing and biofilm formation.Biofilm formation was measured by crystal violet method,and it was found that quercetin could significantly inhibit biofilm formation of ETEC-O139.By measuring the photon number,it was found that quercetin could significantly inhibit the AI-2production of ETEC-O139.The effect of quercetin on the gene expression of biofilm and quorum sensing was detected by RT-PCR,and the results showed that quercetin could significantly inhibit the expression of Lux S,Pfs,Lsr K,Lsr B,Fli C,Csg A,Csg B,Fim A and Rpos.Although quercetin could not significantly inhibit the growth of ETEC-O139,it could inhibit the formation of biofilm by regulating quorum sensing,reduce the pathogenicity of ETEC-O139,and achieve bacteriostatic effect and reduce the probability of drug resistance in this way.According to the above test results,the anti-inflammatory and protective mechanism of quercetin was further studied by establishing an inflammatory injury model of IPEC-J2 cells.By detecting the adhesion of ETEC-O139 to IPEC-J2 cells,it was found that quercetin could significantly inhibit the adhesion of ETEC-O139,which was consistent with the fact that quercetin significantly inhibited the expression of its genes related to adhesion.The protective effect of quercetin on IPEC-J2 cells was also detected by measuring lactate dehydrogenase.The results showed that quercetin could significantly reduce the release of lactate dehydrogenase in IPEC-J2 cells induced by ETEC-O139.The secretion of inflammatory cytokines in IPEC-J2 cells was detected by ELISA and RT-PCR,respectively.Quercetin can significantly inhibit interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)secretion and gene expression,indicating that quercetin has anti-inflammatory effect.Further research results showed that quercetin inhibited the m RNA expression of TLR4,My D88 and NF-κB,indicating that the anti-inflammatory effect of quercetin was achieved by inhibiting the TLR4 signaling pathway and the downstream NF-κB signaling pathway.In conclusion,quercetin affects the transcriptome of ETEC-O139,and studies on its quorum sensing and biofilm formation pathways found that quercetin inhibited the expression of ETEC-O139 quorum sensing and biofilm formation-related genes.In addition,quercetin reduced the adhesion ability of ETEC-139 to IPEC-J2,alleviated the inflammatory damage of IPEC-J2 induced by ETEC-O139,exerted its antiinfection and anti-inflammatory effects,and protected intestinal cells from bacterial infection effect.It provides a theoretical basis for the prevention and treatment of ETEC,and provides new ideas for replacing antibiotics and reducing bacterial resistance,which has important reference significance.