The Role And Mechanism Of Influenza A Virus RHA1 On Inducing Beta Defensins Of Mouse Tracheal Epithelial Cells Based On NF-κB Signaling Pathway

Objective: To explore the effect of recombinant influenza A H1N1 hemagglutinin 1 induce primary mouse tracheal epithelial cells produce BD(β-defensin,BD),and function mechanism based on NF-κB signaling pathway.Methods: Culture mouse primary tracheal epithelial cells,the cells were grouped into control group and experimental group.The experimental group include rHA1 group(200ng/ml),IAV group(2×TCID50),inactivated IAV group,rHA1+IAVgroup,rHA1+inactivated IAV group;Another set inhibition group,the inhibitor PDTC(20μ M)of NF-κB was administered 1 h earlier in the same experimental conditions,and then added to each preparation group.The pathological changes of the cells were observed by HE staining at 4h,8h and 24 h respectively.Collecte cells,taken cell contents,proteins and RNA,detcected the gene and protein expression of mBD2~4,IFN-γ,NF-κB p65,NF-κB P-p65,NF-κB p50,NF-κB P-p50,NF-κB IκB,NF-κB PIκB in each group by qRT-PCR,Western blot and ELISA method respectively.Results: 1.HE staining results showed: After 4h,8h,24 h,the experimental group compared with the control group,cell lesions were found include different size cavitieswere in the cytoplasm,pyknosis in part of nuclear,syncytia etc.2.The results of qRT-PCR showed that the expression of Mbd2~4,IFN-γ and NF-κB p65/p50 gene in the experimental group cells was up-regulated compared with the control group,the expression of Mbd2~4,IFN-γ and NF-κB p65/p50 gene in the rHA1+IAV group was up-regulated compared with the IAV group,the expression of Mbd2~4,IFN-γ and NF-κB p65/p50 gene in the rHA1+ inactivated IAV group was up-regulated compared with the inactivated IAV group.The inhibitor PDTC(20μ M)function cell,compared with the corresponding experimental group without inhibitors,the expression of Mbd2~4,IFN-γ gene in the experimental group cells was down-regulation compared with the control group,the expression of Mbd2~4,IFN-γ gene in the rHA1+IAV group was down-regulation compared with the IAV group,the expression of Mbd2~4,IFN-γ gene in the rHA1+inactivated IAV group was down-regulation compared with the inactivated IAV group.3.The results of Western blot showed that the expression of IFN-γ,NF-κB IκB,NF-κB p65/p50 in the experimental group cells were up-regulated compared with the control group,the expression of IFN-γ,NF-κB IκB,NF-κB p65/p50 in the rHA1+IAV group was up-regulated compared with the IAV group,the expression of IFN-γ,NF-κB IκB,NF-κB p65/p50 in the rHA1+ inactivated IAV group was up-regulated compared with the inactivated IAV group.The inhibitor PDTC(20μ M)function cell,compared with the corresponding experimental group without inhibitors,the expression of IFN-γ in the experimental group cells was down-regulation compared with the control group,the expression of IFN-γ in the rHA1+IAV group was down-regulation compared with the IAV group,the expression of IFN-γ in the rHA1+inactivated IAV group was down-regulation compared with the inactivated IAV group.4.The results of ELISA showed that the expression of mBD2~4 in the experimental group cells was up-regulated compared with the control group,the expression of mBD2~4 in the rHA1+IAV group was up-regulated compared with the IAV group,the expression of mBD2~4 in the rHA1 + inactivated IAV group was up-regulated compared with the inactivated IAV group.The inhibitor PDTC(20μ M)function cell,compared with the corresponding experimental group without inhibitors,the expression of mBD2~4 in the experimental group cells was down-regulation compared with the control group,the expression of mBD2~4 in the rHA1+IAV group was down-regulation compared with the IAV group,the expression of mBD2~4 in the rHA1+inactivated IAV group was down-regulation compared with the inactivated IAV group.Conclusion: 1.The recombinant influenza A virus rHA1 separately or together the living/inactivated Influenza A virus can induce the expression of BD2~4 in mouse tracheal epithelial cells.2.The recombinant influenza A virus rHA1 separately or together the living/inactivated Influenza A virus can induce the expression of BD2~4 in mouse tracheal epithelial cells,which is relate to the NF-κB signaling pathway.