Changes Of MRNA Expression Profile And Inflammatory Signaling Pathway In H9N2 AIV Infected And LPS Stimulated HD11 Cells

H9N2 Avian influenza virus(AIV)is widely distributed throughout the world.It is a common problem in China's poultry industry that chickens are susceptible to E.coli infection after infected with H9N2 AIV.At present,the mechanism of secondary E.coli disease after H9N2 AIV infection is still unclear.In recent years,RNA sequencing technology has been widely used in the study of the pathogenesis of viral infection host cells.This study used high-throughput sequencing technology to detect the changes in mRNA expression profiles of H9N2 AIV infected and LPS-stimulated chicken macrophages(HD11cells).Screening for genes associated with inflammation to further elucidate their relationship to inflammatory signaling pathways.The specific research contents are as follows:(1)Transcriptome analysis of H9N2 AIV infection and LPS stimulation in HD11 cells.The experiment is divided into five groups: Mock,H9N2 AIV was infected with HD11 cells(H9N2 group),H9N2 AIV was infected with HD11 cells and LPS was added to form co-stimulation(co-infection group).H9N2 AIV was first infected with HD11 cells for twenty four hours,then stimulated with LPS for eight hours(secondary infection group).It was found that the cytopathic effects after secondary infection and co-infection were more obvious than H9N2 AIV infection alone.The expression of NS1 protein in the secondary infection group was significantly higher than H9N2 group.The expression of NS1 protein in the co-infected group was significantly lower than the secondary infection group.The above five groups of cells were subjected to RNA sequencing after receiving an RNA sample.The number of up-regulated genes and down-regulated genes screened by H9N2 group,secondary infection group and co-infection group were different.Among them,the co-infection group screened the most differentially expressed genes,447 genes were up-regulated,and 157 genes were down-regulated.Using GO and KEGG Pathway to analyze the differential genes screened,it is shown that these differential genes mainly focus on immune response,inflammatory response,and other pathways.To validate the accuracy of the RNA-Seq technique,we selected six expressed genes associated with inflammation for RT-qPCR,including IFN-?,IL-10,IL-8,TNF-?,IL-12 B,and CXCL12.The study found that RT-qPCR and RNA-Seq results are basically consistent,and the accuracy of sequencing results is preliminarily confirmed,which lays a foundation for the next study on the influence of cellular inflammatory signaling pathway.(2)Changes of inflammatory signaling pathway after H9N2 AIV infection and LPS stimulation in HD11 cells.The key proteins of MAPK and NF-?B signaling pathway were detected by Western blotting.Among them,three key proteins of the MAPK pathway(p38,JNK,ERK)were phosphorylated,and the phosphorylation level of the co-infected group was the most significant;p65 phosphorylation,I?B? degradation,and significant degradation in the co-infected group.It indicated that H9N2 AIV infection and LPS stimulated HD11 cells to activate MAPK and NF-?B signaling pathways.In addition,further studies found that H9N2 AIV infection and LPS stimulated HD11 cells,using p38 inhibitor SB203580 to treat cells,IFN-?,IL-10 significantly changed before and after inhibitor treatment,indicating that IFN-? and IL-10 were promoted by p38 pathway;The cells treated with JNK inhibitor SP600125 showed no significant changes in inflammation-related genes before and after treatment,indicating that IFN-?,IL-10,CXCL12,TNF-? and IL-8 did not pass through the JNK pathway;Treatment of cells with the ERK inhibitor SCH772984,IL-8 significantly changed before and after treatment,indicating that the ERK pathway inhibits IL-8 secretion;IFN-?,IL-10,CXCL12,TNF-? and IL-8 were significantly changed before and after treatment with NF-?B inhibitor BAY11-7082,indicating that indicating that IFN-?,CXCL12,TNF-? and IL-10 were promoted by NF-?B signaling pathway,and inhibit IL-8 secretion.Thus elucidate the relationship between IFN-?,IL-8,IL-10,TNF-?,CXCL12,IL-12 B and MAPK or NF-?B signaling pathways.In summary,this study screened the intracellular genes differentially expressed in H9N2 AIV infected and LPS-stimulated HD11 cells by RNA sequencing technology.These differential genes help to resolve the relationship between H9N2 AIV infection and E.coli infection.Further studies have shown that MAPK and NF-?B pathways regulate the inflammation-related genes in the process of secondary infection and co-infection.The results provide useful experimental data for revealing the molecular mechanism of secondary E.coli disease after chicken infection with H9N2 AIV Provide a scientific basis for the clinical development of the continuous epidemic of H9N2 AIV and the prevention and treatment strategies of secondary E.coli infection.