The Role Of Autophagy Induced Macrophage In Brucella Via AMPK/p70s6k Signaling Pathway

Brucellosis is one of the most widespread zoonoses in the world caused by several species of the genus brucella. Brucella is a gram-negative and facultative intracellular bacterial pathogen. Macrophage was one of important target cells for its present and replicates, brucella intracellular survival is associated with inhibition of host cell apoptosis and advance cell autophagy. At present, the research has shown that AMPK / p70s6 k signaling pathway is not only associated with a variety of cellular activities for apoptosis and autophagy and so on, but also closely related to the survival and reproduction of pathogen in the cellular.Objects: To detect whether Brucella melitensis 16M(16M) can activate AMPK/p70s6 k pathway of murine macrophages RAW264.7(RAW264.7) and promote cell autophagy through the experiment based on 16 M infection model in vitro, and to identify the persistent infection of brucella in the host cell is closely related to AMPK / p70s6 k signaling pathways.Methods: 1. Establish the experimental model of brucella melitensis 16M(16M) infected murine macrophages RAW264.7(RAW264.7) in vitro, using Western blot detected the protein levels of p-AMPK(Thr172) and p-p70s6k( Thr389) in different time.2. The RAW264.7 were treated with inhibitor Compound C of AMPK for 1 h, then the cells were infected with 16M; Using CFU we detected the intracellu Lar growth of 16 M was determined at 4h, 8h, 12 h, 24 h and 48 h post-infection; Using Western Blot we detected the expression level of p-AMPK(Thr172) and p-p70s6k(Thr389) which treated by inhibitors Compound C after the 16 M infection.3. The RAW264.7 were treated with inhibitor Compound C of AMPK for 1 h, then the cells were infected with 16M; Using real-time quantitative PCR and Western blot to detect the m RNA and protein levels of autophagy-related genes LC3-II/LC3-I, Beclin1 and ULK1.4. Three si RNA fragments for different sites on AMPKα2 gene were designed with small interfering RNAs software, and then these si RNA fragments was constructed by the company.The specific si RNA was transferred into RAW264.7 by si RNA-mate mediated transfection.The cells were collected after post-transferred 24 h, and using q RT-PCR and Western blot to detect the m RNA and protein levels of AMPKα2. Then the silence effects of three pairs of specific si RNA fragments corresponding to different sites on AMPKα2 were analyzed to screen out the AMPKα2-si RNA fragment with the best silence effect.5. The best silence effect of si RNA fragment was transferred into RAW264.7 cells, and after post-transferred 6h used 16 M to infect the cells. The cells which were not transferred AMPKα2-si RNA fragment and only transferred with negative control fragment were seen as control group. Then using CFU to detect the intracellular growth of 16 M and using real-time quantitative PCR we detected the m RNA levels of autophagy-related genes Beclin1 and ULK1.Results: 1.The expression of p-AMPK(Thr172) was highest and significantly higher than the other groups at 1 h post-infection(P<0.05), which returned to normal and had no significant difference compared with the control group at 8h, 12 h and 24 h post-infection(P>0.05).Howere, the expression of p-p70s6k(Thr389) was lower than the control group(P <0.05).2. The number of intracellular 16 M treated with Compound C was significantly lower than the control cells at all observation times(P<0.05).3. The expression level of p-AMPK(Thr172), Beclin1 and ULK1 were inhibited by Compound C, but the expression of p-p70s6k(Thr389) was higher than those cells which were treated by 16M(P <0.05). Moreover, the m RNA expressions of Beclin1 and ULK1 which were treated by Compound C were both significantly lower than 16 M at 12 h and 24h(P <0.05).4. The specific si RNA fragments constructed could effectively reduce the levels of AMPK m RNA and protein in RAW264.7 and the silence efficiency was higher than those in normal group, si RNA-mate group and negative group, showing statistically significant differences(P<0.05). Among the three pairs of si RNA fragments corresponding to different sites,AMPKα2-865-si RNA demonstrated the strongest inhibiting effect on AMPK m RNA and protein.5. The number of intracellular 16 M in silencing AMPKα2 gene cell group was significantly lower than the control group and the NC cells group at 12 h and 24h(P<0.01); And at 12 h and24h the expression levels of proteins p-AMPK(Thr172), LC3-II/LC3-I, Beclin1 and ULK1 in silencing AMPKα2 gene cell group were significantly lower than 16 M cells group(P<0.01),while the expression level of protein p-p70s6k(Thr389) was higher than 16 M cells group(P<0.01), and the m RNA relative expression of Beclin1 and ULK1 were significantly lower than 16 M cells group(P <0.01).Conclusions: 1. Brucella could activate AMPK/p70s6 k signaling pathway.2. The inhibitor Compound C could significantly inhibit the intracellular growth of 16 M.3. The inhibitor Compound C could significantly inhibit the 16M-mediated autophagy.4. After AMPKα2 gene silenced, the intracellular growth of 16 M could significantly inhibit as well as inhibit the 16M-mediated autophagy.