Fusobacterium Necrophorum Induced Autophagy In RAW264.7 Through AMPK-mTOR Signaling Pathway

Fusobacterium necrophorum(Fn),a Gram-negative,strict anaerobic bacteria without spore,most of them are long filaments and short rods,Fn can induce liver abscess and hoof rot in cattle and sheep,human Lemierre’s syndrome and other necrotic diseases.Autophagy is an important process of degrading damaged proteins and organelles for recycling.Autophagy has been found to play an important role in bacterial infection.Whether Fn can induce autophagy and what role autophagy plays in Fn infection have not been reported.Therefore,in this study,Fn and mouse macrophage system(RAW264.7)were used as research objects to explore the signaling pathway of autophagy induced by Fn and the role of autophagy in Fn infection,provide a theoretical basis for the pathogenic mechanism of Fn.In order to study the effect of Fn on the proliferation of RAW264.7 cells,the Fn were infected with MOI of 0,0.5: 1,1: 1,10: 1,50: 1 and 100: 1.The proliferation of RAW264.7 cells was detected by Ed U method after 0.5 h,1 h,2 h and 4 h.The results showed that necrotic bacilli inhibited macrophage proliferation in a time and dose dependent manner.In order to determine the optimal multiplicity of infection for Fn induced autophagy in RAW264.7 cells,RAW264.7cells were infected with Fn at MOI of 0,1: 1,10: 1,and 50: 1,and the expression of LC3 and P62 proteins was detected by Western blot.The results showed that the protein expression of LC3-Ⅱ was significantly regulated up(P<0.01)and the protein expression of P62 was significantly regulated down(P<0.05)when the multiplicity of infection was 10: 1.Next,in order to determine the autophagy and signaling pathway of RAW264.7 cells induced by Fn,RAW264.7 cells were infected with Fn at MOI of 0 and 10:1 for 1 h,2 h,and 4 h.The effects of Fn on autophagy of RAW264.7 cells were investigated by MDC staining,transmission electron microscopy,q RT-PCR,and Western blot.To determine the condition and signaling pathway of autophagy induced by Fn.MDC results showed that the fluorescence intensity gradually increased with the increase of infection time.Transmission electron microscopy results showed that compared with the control group,autophagic lysosomes were observed in the infection group.The results of qRT-PCR showed that there was no significant change in the relative transcription levels of Beclin-1,LC3,ATG5 and ATG7 mRNA at 1 h after infection.The relative transcription levels of Beclin-1,LC3,ATG5 and ATG7 mRNA were regulated up at 2 h and 4 h after infection(P<0.01).The relative transcription level of P62 mRNA showed a downward trend at different infection time(P<0.01).The results of Western blot showed that the expression of LC3-Ⅱ protein was regulated up at different infection time(P<0.01),and the expression of P62 protein was regulated down at different infection time(P<0.01).The protein expression levels of ATG5,ATG7 and Beclin-1 did not change significantly at 1 h after infection,and the protein expression levels of ATG5,ATG7 and Beclin-1 increased at 2 h and 4 h after infection(P<0.01).With the addition of RAPA and CQ,the protein expression of LC3-Ⅱ showed a regulated up(P<0.01).In the case of adding RAPA,the protein expression of P62 showed a regulated down(P<0.05),while in the case of adding CQ,the protein expression of P62 showed an upward trend(P<0.05).The protein expression levels of p-AMPK and p-ULK1 did not change significantly at 1 h after infection,and the protein expression levels of p-AMPK and p-ULK1 increased at 2 h and 4 h after infection(P<0.01).There was no significant change in the protein expression of p-mTOR at 1 h and 2 h after infection,and the protein expression of p-mTOR was regulated down at 4 h after infection(P<0.05).Finally,RAW264.7 cells were infected with Fn at MOI of 0 and 10: 1,and the bacterial load was detected by colony counting.The results showed that the bacterial load of the experimental group was significantly lower than that of the control group(P<0.05).Tn summary,Fn promoted autophagy in RAW264.7 cells,through AMPK-mTOR signaling pathway to promote autophagy,and its induced autophagy can limit the proliferation and replication of Fn,providing a theoretical basis for the pathogenesis of Fn.