| Newcastle Disease(ND)is an acute,severe and highly contagious infectious disease characterized by Newcastle Disease Virus(NDV),characterized by avian respiratory tract and gastrointestinal mucosal hemorrhage.NDV can cause the tissues different degrees of exudative inflammation.NLRP3 Inflammation is a key immunoregulatory factor in virus-induced Inflammation response.NLRP3 Inflammation plays an important role in the activation of caspase-1 and IL-1? maturation and secretion process.And mainly from the following aspects to introduce:1.Chicken NLRP3 Inflammation research systemIn this study,we first established the study system of chicken NLRP3 Inflammation.Firstly,the recombinant protein of caspase-1 and IL-1? was expressed by prokaryotic expression system of Escherichia coli,and then the recombinant proteins were purified by the method of gelatin reclamation.The polyclonal antibody was able to detect the corresponding protein at 1 : 500 dilution,and proved that it could be used in the study of NLRP3 Inflammation body.The establishment of NDV-activated NLRP3 Inflammation body was established,which laid the foundation for the futher study.2.Effects of different virulence NDV infected SPF chickens on NLRP3 signaling pathwayIn this study,we used the rGM strain and the IGM strain of the strong and weak NDV strain screened by the reverse genetic system to observe the inflammation caused by different strains,and used qPCR to analyzed the effect on the expression of proinflammation cytokines IL-1? and IL-18 transcription and NLRP3 Inflammation expression.The results showed that the expression of NLRP3 in different tissues wasconsistent with that of proInflammation cytokines IL-1? and IL-18,and the expression of NLRP3,IL-1? and IL-18 in rGM group were significantly higher in lung,thymus and spleen,and higher than those in the control group.The expression of IL-1?,IL-18 and NLRP3 in the lungs and brain was significantly up-regulated in the IGM group,that showed a very obvioust difference from the mock group,while the expression level in the bursa of Fabricius,thymus and spleen was low.3.The effect of DF-1 cells infected with NDV rGM on NLRP3 signaling pathwayTo investigate the relationship between NLRP3 and NDV inflammation,we investigated the expression of IL-1?,IL-18 and NLRP3 in DF-1 cells after NDV infection.IL-1?,IL-18 and caspase-1 were up-regulated and the genetic expression of NLRP3 was up-regulated 1.8 times.The expression of NLRP3 was dose-dependent in infected cells with NDV.The expression of caspase-1 and IL-1? was down-regulated significantly after treated with si-RNA-NLRP3.Due to the NLRP3 overexpression,the expression of caspase-1 and IL-1? were significantly up-regulated.Suppression or overexpression of the NLRP3 gene did not affect the replication and proliferation of NDV.4.NLRP3 Inflammation Regulates the Signal Pathway of Inflammatory Response Induced by NDV InfectionIn order to further study the regulatory mechanism of NLRP3 inflammatory in the inflammatory response of NDV,NF-?B and S1RP1 signaling pathway related to the expression of IL-1? were selected.In this study,DF-1 cells treated with si-RNA-NLRP3 were infected with NDV virulent rGM strain.There was no significant difference in the expression of S1PR1 and P65 mRNA.Treated with S1PR1 inhibitor W146 and infected with NDV virulent rGM strain.And the expression of NLRP3 and P65 mRNA was significantly decreased.Overexpressing S1PR1 was treated with NDV virulent rGM strain.The expression of NLRP3 and P65 mRNA was significantly increased.After treatment with si-RNA-P65,the expression level of S1PR1 mRNA was not significantly different,but the expression level of NLRP3 mRNA was significantly decreased.Therefore,the preliminary results showed that the DRV-rGM strain could activate S1PR1,NF-?B and NLRP3 signaling pathway after DF-1 cells infection,and the regulation of NLRP3 Inflammation was dependent on NF-?B and S1PR1 signaling pathway.5.The Inflammation of Dendritic Cells from chicken bone marrow induced by different virulence NDVIn order to study the inflammatory response of NDV infection,we studied the dendritic cells(DCs)of chicken bone marrow as a model to study the inflammatory response of NDV.The DCs were induced by human GM-CSF and IL-4 in vitro.The NDV strain rGM strain and IGM strain were inoculated to detect the replication of NDV and the transcriptional expression of NLRP3.The results showed that the proliferation of NDV in DCs was similar to that in DF-1 cells,but the transcriptional expression of S1PR1,NLRP3 and P65 after NDV infection was different from that of DF-1.Based on the dynamic changes of virus infection and immune system in NDV infection,this study was conducted to study the molecular mechanism of NDV infection leading to exudative inflammation of the body.It was found that NDV infection could promote the production of IL-1? by activating NLRP3 inflammation body and enrich the content of NDV pathogenesis,and provide scientific basis for the prevention and control of ND in clinical practice. |
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