Study On The Regulation Of Immune Cell Apoptosis And Inflammatory Response By Fusobacterium Necrophorum

Fusobacterium necrophorum(Fn),a non-spore,strictly anaerobic,multi morphological Gram-negative bacterium,which can induce a variety of diseases including hoof rot of cattle and sheep,liver abscess and human Lemierre?s syndrome.Fn can induce apoptosis and inflammation in a variety of cells,among which,neutrophils and macrophages as important defense cells also included,but the mechanism is still unclear.In order to explore the mechanism of apoptosis and production of inflammatory factors induced by Fn,In this studied,the immune response induced by Fn in sheep neutrophils and macrophages(RAW246.7)was explored,which provided the basis for studying the mechanism of Fn infection.This experiment mainly includes four parts:(1)The effect on proliferation of macrophages caused by Fn.Macrophages were infected with the plural numbers of MOI 0,50:1,100:1,200:1,500:1,1000:1 for 2 h,4 h and 6 h,respectively.The results showed that the proliferation of macrophages was inhibited in a dose and time-dependent manner by Fn,and 100:1 MOI was selected as the treatment condition for follow-up test.(2)Apoptosis of neutrophils and macrophages induced by Fn.Hoechst staining,DNA ladder and flow cytometry were used to detect the apoptosis of neutrophils and macrophages in sheep peripheral blood infected with Fn at MOI of 0 and 100:1 for 2 h,4 h and 6 h.DNA Ladder analysis showed that the fluorescence staining of macrophage apoptosis was enhanced after treatment with Fn,and DNA showed "ladder" band;flow cytometry indicated that Fn promoted apoptosis,and the apoptosis was the most significant at 4 h.(3)Transcriptome analysis of the effect of Fn on sheep neutrophils.Neutrophils were treated with Fn for 4 h for transcriptome analysis,The results showed that Fn induce apoptosis and production of inflammatory factors in sheep neutrophils by NF-? B and mitochondrial signaling pathways.(4)Regulation on apoptosis and inflammatory gene expression by Fn.The expression of apoptosis related Bcl-2,Bax,AIF,Cytc,Caspase-3,and inflammatory related TNF-?,IL-1?,IL-6 were detected by q RT-PCR and Western Blot,which proved that Fn could promote the apoptosis of immune cells and the expression of inflammatory factors from gene and protein levels.This study demonstrated that Fn advances apoptosis of neutrophils and macrophages,and also promotes immune cell apoptosis and inflammatory response by regulating Bcl-2,Bax,AIF,Cytc,Caspase-3,TNF-?,IL-1? and IL-6,which lays a foundation for the study of pathogenic mechanism of Fn.