| Equine β-defensin-1(eBD-1)is a member of the defensin families,and it widely distributed in Equus.In previous,the prokaryotic expression of eBD-1 was carried out in our research,and the result showed that eBD-1 had inhibited effect on the growth of Staphylococcus aureus(S.aureus)and Escherichia coli(E.coli).However,there was no reported whether eBD-1 was involved innate immune responses.In this study,it was further explored that the bacteriostatic effect of eBD-1-ELP,and demonstrated the immune mechanism of eBD-1-ELP on mouse mononuclear macrophage.The contents of the study were following:(1)To further study the bacteriostatic effect of eBD-1-ELP,PCR,bienzymatic digestion and sequencing methods were used to identify eBD-1-ELP sequence in E.coli expression system.The eBD-1-ELP expression conditions were optimized in OD value and Western blot methods.The minimum inhibition concentration of the eBD-1-ELP recombination protein for E.coli(ATCC 25922)and S.aureus(ATCC 25923)were calculated by bacterial colony count method,and the inhibition effective of eBD-1-ELP recombination protein for Enteropathogenic Escherichia coli(EPEC)and S.aureus(SA113)were calculated also by the same method.In addition,treated eBD-1-ELP into EPEC-infected BALB/c mice model was used in the study,and cytokines secretion in mice blood were measured by ELISA,and the damages of duodenum,jejunum,cecum were investigated by H.E staining after 15 days.The results showed that eBD-1-ELP recombination protein with a better phase transition at35 ℃,and eBD-1-ELP expression in a high level after twice inverse transition cycling(ITC).It showed that the minimum inhibition concentration of the eBD-1-ELP recombination protein for ATCC 25922 and ATCC 25923 were 33.3 μg/m L and 25.0 μg/m L,respectively.Further,IL-6 and TNF-α secretion in mice blood and the damages of duodenum,jejunum and cecum were markedly decrease when treated eBD-1-ELP in EPEC-infected mice after15 days.(2)In order to clarify the role of eBD-1-ELP in macrophage innate immune responses.SA113-infected mouse mononuclear macrophage(J774A.1)was used as a main research subject in this study.The cytokines expression was detected by the method of ELISA and q PCR,and bacterial colony count method and fluorescent staining were used to check J774 A.1 phagocytosis SA113 when treated eBD-1-ELP.The results showed that eBD-1-ELP promoted cytokines expression in SA113-infected J774 A.1cells,and enhanced the effect of J774 A.1 phagocytosis SA113.(3)To explore the regulation mechanism of eBD-1-ELP to innate immune responses on macrophage.SA113-infected J774 A.1 cells was used as a main research subject in this study,and the level of phosphorylation of spleen tyrosine kinase(Syk),focal adhesion kinase(FAK),PI-3K-AKT and IκB-α were measured by Western blot.The Western blot and immunofluorescence methods were also used to detected FAK,paxillin,vinculin expression.In addition,PI-3K inhibitor was added after eBD-1-ELP treated in SA113-infected J774 A.1cells.The level of phosphorylation of AKT and IκB-α were investigated by Western blot,and the cytokines expression was measured by q PCR.Moreover,in this study,FAK small interfering RNA transfected into J774 A.1 cells before eBD-1-ELP treated SA113-infected.The Western blot method was used to detect the expression of FAK,paxillin and vinculin.The effective of J774 A.1 phagocytosis SA113 was investigated by bacteria count method and fluorescent staining method.The results showed that the level of phosphorylation of Syk,FAK,PI-3K-AKT and IκB-α was increased in eBD-1-ELP treated SA113-infected J774 A.1cells.However,the level of phosphorylation of PI-3K-AKT and IκB-α and cytokines expression were down-regulated by PI-3K inhibitor.Further,after SA113 and eBD-1-ELP were added,the FAK,paxillin and vinculin expression and the ability of J774 A.1phagocytosis SA113 were significantly decreased,when J774 A.1 cells transfected FAK small interfering RNA.The study suggested that eBD-1-ELP played an inhibition role for bacteria growth,either EPEC-infected mice or cultured bacterium in vitro.Moreover,eBD-1-ELP upregulated cytokines secretion in SA113-infected J774 A.1 cells via PI-3K-AKT-NF-κB pathway.In addition,eBD-1-ELP promoted the ability of J774 A.1 cells phagocytosis SA113 via FAK-paxillin-vinculin pathway. |
PDF Full Text Request