| Chicken colibacillosis is a bacterial infectious disease caused by certain pathogenic strains of Escherichia coli.Avian pathogenic Escherichia coli(APEC)causes a wide range of localized or systemic diseases in avian species with a variety of clinical signs,including pericarditis,perihepatitis,septicemia,air sacculitis and meningitis,APEC can breach the blood-air barrier to induce septicemia.Chicken Type II pneumocytes play an important role of maintaining the function of blood–air barrier.Previously,the chicken type II pneumocytes have been isolated and purified.It has the characteristics of epithelial cells examined by alkaline phosphatase and electron microscopy.In order to further determine the purity of chicken type II pneumocytes,we used the corresponding antibodies CD74,TTF1,CK19,CK18,Vimentin by flow cytometry and immunofluorescence.Flow cytometry results showed that chicken type II pneumocytes accounted for 96% of the number of cells,fibroblasts accounted for 0.7%,non-lung cells accounted for about 2%.Cell immunofluorescence assay revealed that,except for vimentin,the specific immunofluorescence of the other antibody staining can be seen.The high purity of cells laid the foundation for the mechanism of rutin inhibiting the invasion of E.coli to chicken type II pneumocytes.APEC contains many virulence factors,including iron uptake related genes(IucD,fyuA),type 1 pili(Fim A,Fim B,FimC),curly pili A(CsgA,CsgB),outer membrane protein A(OmpA)and so on.These virulence factors are closely related to bacterial iron uptake,metabolism,adhesion and invasion.The extracellularly aggregated signal molecule AI-2 can regulate the transcriptional level of the virulence factors through the quorum sensing system,which affects the pathogenicity of bacteria.In terms of bacteria,we investigated the effects of rutin on quorum sensing system of Escherichia coli.It was found that when APEC-O78 was treated with 25-50?g/m L of rutin for 4h,the related virulence genes(Rpos?H-NS?iucD?fyuA?CsgA?CsgB?Flic?Lsr B?LsrK?Luxs?Pfs)were decreased significantly(P <0.05).Rutin affected the expression of bacterial survival stress gene by reducing the secretion of AI-2 to interfere with the quorum sensing,and rutin reduced the APEC-O78 biofilm formation ability.In terms of cell,the RNA-seq was used to investigate the pathogenesis of Escherichia coli invading chicken type II pneumocytes and the interference mechanism of rutin.First,we determined the optimal sample treatment method: 50?g/mL rutin pretreatment with cells for 4 h,then CPII cells were infected with APEC-O78 for 4h.The results showed that there were 1390 genes with significant differences between APEC-O78 and non-infected groups(P <0.05).There were 587 up-regulated genes and 803 down-regulated genes.After analysis of APEC-O78 infection,the RNA-seq results shows the immune response involved NF-kappa B signaling pathway,apoptosis,inflammatory response,cytokine-cytokine receptor interaction,and Toll-like receptor signaling pathway.CP II cells were pretreated with rutin 50?g / m L for 4h,and after being washed,CPII cells was infected with APEC-O78 for 4h.A total of 222 genes with significant differences(P <0.05)were analyzed,there were 59 down-regulated genes and 163 up-regulated genes,the rutin may improved CP II cells involves E.coli infection,phagocytosis,close attachment and glycolysis/ gluconeogenesis,glycosaminoglycan biosynthesis,pentose phosphate pathway,etc.In summary,the results shows that rutin inhibited the secretion of AI-2 to interfere with the quorum sensing,reduced the expression of virulence genes,and decreased the APEC-O78 biofilm formation ability,thereby inhibiting the damage of E.coli to CP II cells.In terms of cell,RNA-seq results showed that rutin may improved CP II cells involves E.coli infection,phagocytosis,close attachment and glycolysis/gluconeogenesis,glycosaminoglycan biosynthesis,pentose phosphate pathway,etc.These provide the evidence for the application of rutin in the clinical and other biological fields of veterinary medicine. |
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