Identification And Functional Study Of Genes Related To Stress-induced Immunosuppression In Chicken Thymus

With the rapid development of scale,intensification and industrialization of poultry farming,the losses caused by stress response are becoming more and more serious.Therefore,the research and prevention of the stress response should be paid great attention.Thymus is the site of the differentiation,development and maturation of poultry T cells,which plays an important role in immune regulation.Immunosuppression caused by stress can affect its immune function through gene changes in the thymus.At present,there are few and not enough in-depth studies on the molecular regulation mechanism of stress affecting poultry immune function.In this study,the thymus of the previously constructed corticosterone-induced Gushi chicks stress model group and the control group were used as the research objects.RNA-seq technology was used to identify and analyze the differentially expressed m RNA and enrichment pathways in the thymus of the stress model group and the control group.The stress-induced immunosuppression related genes in chicken thymus were screened out,and the candidate gene HSPA8 was discussed in depth to study the immune regulation function and mechanism of this gene at the individual and cellular levels.At the individual level,the model of beak trimming stress and heat stress was established to study the effects of beak trimming stress 1d,3d,5d and 7d and continuous heat stress 28 d on serum biochemical indices and immune-related indices of Gushi chickens,and to verify the changes of HSPA8 expression in thymus of chickens at different time points after beak trimming and continuous heat stress;based on the cloned sequence of the CDS region of the HSPA8 gene,the biological characteristics and structural characteristics of the gene were analyzed by bioinformatics methods;at the cellular level,the effects of HSPA8 on the proliferation,apoptosis and immune function of HD11 cells were studied by overexpression and interference of HSPA8 in HD11 cell lines and co-treatment with LPS,and the role of HSPA8 in the immune response of HD11 cells was discussed.The main test contents and results are as follows:1.Transcriptomic study on the thymus of corticosterone-treated chicksRNA-seq results showed that there were 101 differentially expressed genes in the chick thymus of the corticosterone-treated group and the control group,including 44 up-regulated genes and 57 down-regulated genes.The results of integrated analysis showed that XCL1,HSPA8,DMB1 and BAG3 were strongly correlated with immunity and stress,and these genes may have a potential regulatory role in the process of stress affecting the immune function of chicken thymus.Finally,the candidate gene HSPA8 was determined as the object of follow-up study,and the expression of this gene in the thymus of the corticosterone-treated group was significantly lower than that of the control group(P < 0.05).2.Validation of the candidate gene HSPA8 in different stress modelsSerum biochemical analysis and ELISA results showed that both beak trimming stress and continuous heat stress could significantly promote the increase of chicken serum CORT content and the release of IL-1β,IL-6 and TNF-α(P < 0.05),significantly decreased CD3+ and Ig G content(P < 0.05).qRT-PCR results showed that the expression of IL-1β,IL-6,TNF-α and IL-8 in chichen thymus were significantly increased on the 1d(P < 0.05),significantly decreased on the 5d(P < 0.05),and significantly increased in the continuous heat stress group(P < 0.05);the expression of pro-apoptotic genes increased,and the anti-apoptotic genes decreased;the expression of HSPA8 in thymus was significantly increased on the 1d and 3d after beak trimming(P < 0.05),significantly decreased on the 5d(P < 0.05),had no significant change on the 7d(P < 0.05),and significantly decreased in the continuous heat stress group(P < 0.05).3.Analysis of bioinformatics and tissue expression of chicken HSPA8The sequence of CDS region of the chicken HSPA8 gene is 1941 bp,encodes 646 amino acids.Bioinformatics analysis results showed that chicken HSPA8 protein is a negatively charged,stable and hydrophilic protein,without signal peptide region and transmembrane domain,has multiple phosphorylation and glycosylation modification sites;the protein mainly functions in the nucleus,it has two conserved domains and forms an interaction network with DNAJC5,HSPB1,DNAJA1 and GAK,etc.;the chicken HSPA8 gene has the closest relationship with turkey and the farthest relationship with lower fish.Tissue expression profile results showed that HSAP8 was widely expressed in tissues and organs of chicks,with the highest expression in pancreas and lung,and the lowest expression in leg muscle and and chest muscle.4.To study the gene function of HSPA8 at the cellular levelCCK-8,EdU,flow cytometry and qRT-PCR results showed that overexpression of HSPA8 could significantly increase the activity of HD11 cells(P < 0.05),significantly promote cell proliferation(P < 0.05),and significantly inhibit cell apoptosis(P < 0.05),the interference was the opposite;overexpression/interference of HSPA8 and LPS cotreatment experiments showed that overexpression of HSPA8 could further promote the expression of LPS-induced inflammatory cytokines,while interference was the opposite;overexpression of HSPA8 could promote the activation of TLR4/My D88/NFk B signaling pathway in HD11 cells,and significantly up-regulate the expression of key factors ERK,JNK and P38 genes in the MAPK signaling pathway(P < 0.05),while interference was the opposite.In conclusion,the expression of HSPA8 in the thymus of the corticosteroneinduced Gushi chicken stress model group was significantly lower than that of the control group;the expression of HSPA8 in the thymus of chicks was significantly increased on the 1d and 3d after beak trimming,significantly decreased on the 5d and had no significant change on the 7d,and significantly increased on the 28 d of continuous heat stress;HSPA8 could promote the proliferation of HD11 cells,inhibit cell apoptosis,and participate in regulating the immune response of HD11 cells,and promote the expression of LPS-induced inflammatory cytokines;HSPA8 may promote the activation of TLR4/My D88/NF-k B and MAPK signaling pathway in HD11 cells.The above results indicate that HSPA8 is involved in regulating the stress immune response of poultry and can be used as a reliable indicator to reflect whether the poultry are in the state of stress immunosuppression.The results of this study provide a theoretical basis and reference for revealing the molecular regulatory mechanism of stress affecting poultry immune function.