| Brucellosis is a zoonotic infectious disease caused by Brucella. Brucella canprevent fusion with lysosomes, and inhibit apoptosis and promote autophagy withinRAW264.7 cells, resulting in Brucella intracellular survival and growth within phagocyticcells. Brucella can cause multiple organ damage and chronic infection in humans and animals,and bring a serious threat for the harmonious development of human health and livestock.The PI3K/Akt signaling pathway could regulate a variety of cellular activities and biologicaleffects of cell metabolism, gene transcription, cell migration movement, such as the cell cycleand proliferation and apoptosis. However, studies have shown that PI3K/Akt signalingpathway involved in intracellular survival and growth of viruses within cells. It ispresently unclear that whether this pathway involves Brucella intracellular survival andgrowth within phagocytic cells. Therefore, the study on the effect of PI3K/Akt pathway onBrucella intracellular survival and growth within phagocytic cells is of major significancefor revealing the pathogenic mechanism of Brucella, and providing scientific basis forexploring candidate drug targets.Objects:(1) We detect whether Brucella melitensis 16M(16M) can activate PI3K/Aktpathway of RAW264.7 macrophages.(2) We detect the effect of inhibition of the PI3K/Aktpathway on the survival of 16 M in vivo and in vitro.(3) We detect the effect of inhibition ofthe PI3K/Akt pathway on the 16M-dependent apoptosis of RAW264.7 macrophages.(4) Wedetect the effect of inhibition of the PI3K/Akt pathway on the 16M-dependent autophagy ofRAW264.7 macrophages.(5) We detect the function of PI3 K catalytic subunit p110α in thecourse of 16 M infecting RAW264.7 macrophages. Our study is useful for demonstrating themolecular mechanisms regulating persistent 16 M infection and laying a foundation for thedevelopment of new drug and prevention of brucellosis and breeding of anti-brucellosislivestock.Methods:(1) We dectected the protein levels of p-Akt(S473) or p-Akt(T308) usingwestern blot: we observed whether PI3K/Akt signaling pathway was activated by 16 M, otherstrains or lipopolysaccharide(LPS), and whether the activation of PI3K/Akt signalingpathway was related to the infection time and MOI of 16 M, and the inhibition effect of16M-dependent PI3K/Akt pathway was inhibited by LY.(2) RAW264.7 macrophages weretreated with inhibitor LY for 1 h, then, the cells were infected with 16 M, cells not treated withLY as a control group. Brucella CFU were determined at 30 min, 45 min, 4 h, 12 h or 24 hpostinfection. The production of IFN-γ, IL-12p70, or TNF-α in cell-culture supernatants weredetected using ELISA. Six-to-eight-weeks-old female BALB/c mice(n=10) were injected-intraperitoneally with LY(20 mg/kg/day) and continued until the end of the experiment. Weobserved the mortality rates in mice infected with LD100(4.6×108CFU/0.2m L) or LD50(6.3×107 CFU/0.2m L) after giving LY for 5 days. The mice were infected with 1×106CFU/0.2m L dose of 16 M. The spleen and liver of mice were exteriorized and the CFU of16 M were calculated and pathological changes were observed using biopsy technique.(3)The RAW264.7 macrophages were treated with LY for 1 h and then infected with 16 M. Forthe control group, cells were not treated with LY. The activities of caspase-3,8,9 weredetected using assay kit. The expression of apoptosis-related genes were detected usingreal-time quantitative PCR and Western blot. The apoptosis rates were detected using flowcytometry.(4) Cells were transfected with lentiviral packaging plasmid with Plex-MSC-EGF-P-LC3 and then treated with LY for 1 h and then infected with 16 M for 12 h and 24 h, thep EGFP-LC3 green punctate dots and co-localization of p EGFP-LC3 with lysosomes wereanalyzed by confocal microscopy. The number of autophagic vesicles containing 16 M wereobserved under transmission electron microscopy.(5) According to the RNAi designprinciples, four specific positive si RNA duplexes targeted p110α gene were designed byClontech, and one negative si RNA duplexes was taken as the control. The Lentiviral vectors-p Lenti Lox-si RNA were constructed and RAW264.7 cells were transduced with p Lenti Lox-si RNA plasmid. The m RNA expression of p110α were detected by real-time PCR to screenthe best p Lenti Lox-si RNA plasmid.Results:(1) The protein expressions of p-Akt(S473) and p-Akt(T308) were bothhighest and significantly higher than other groups at 1 h post-infection(P<0.05), whichreturned to normal and had no significant difference compared with the control group at 8 h,12 h and 24 h post-infection(P>0.05). The protein expressions of p-Akt(S473) and p-Akt(T308) for cells infected with rough type strains(RB51, M5Δpgm) were significantly lowerthan those for cells infected with smooth type strains(M5,16 M and S2308)(P<0.01). Theprotein expressions of p-Akt(S473) and p-Akt(T308) were inhibited by LY a dose-dependentmanner.(2) The number of bacteria inside cells treated with LY was significantly lower thanthat of control cells at all observation times(P<0.05). At a dose of LD100 and LD50 on day 10 postinfection, the survival rates of mice treated with 16M-LY were 30% and 60%,respectively, and those of mice treated with 16 M were 0 and 30%, respectively. Serum levelsof IFN-γ, IL-12p70 and TNF-α from mice treated with 16M-LY were significantly higherthan those of control mice treated with 16M(P<0.01). The percentage of CD8+T cells in micetreated with 16M-LY were significantly higher than those of control mice treated with 16M(P<0.01). The pathological changes in mice treated with 16M-LY were fewer than those ofcontrol mice treated with 16 M.(3) At 4 h, 12 h, and 24 h post-infection, the caspase-3 active-ty(A405 value) in cells treated with 16M-LY was significantly higher than that of controlcells treated with 16M(P<0.05), and with a similar trend for the caspase-9 activity(A405 value). The expressions of Bax m RNA and proteins of cleaved PARP and Bax in cellstreated with 16M-LY were significantly higher than that of control cells treated with 16M(P<0.01). The apoptosis rates of cells treated with 16M-LY were significantly higher than thatof control cells treated with 16M(P<0.01).(4) The p EGFP-LC3 green punctate dots in cellstreated with 16M-LY were significantly fewer than those of control cells treated with 16M(P<0.01). The m RNA expressions and protein levels of Beclin1 and ULK1 and conversionrates of a fraction of LC3-I into LC3-II and autophagic vacuoles in cells treated with 16M-LYwere significantly lower than those of control cells treated with 16M(P<0.01).(5) Thenumber of 16 M were not significantly different within macrophages following the p110αgene silencing compared with that of control cells at 30 min, 45 min, and 4 h post-infection(P>0.05), which had a significant difference at 12 h and 24 h post-infection(P<0.05). At 12 hand 24 h post-infection, compared with control cells, the activity of caspase-3 and proteinsexpression of cleaved PARP, Bax, and Cytosolic Cyt-c were significantly increased(P<0.01),and m RNA expression levels of Bcl-2 were significantly decreased(P<0.01). The levels ofIL-12p70 and TNF-α were significantly increased(P<0.01).Conclusions:(1) Brucella and LPS can activate PI3K/Akt signaling pathway.(2) The16 M survival was significantly reduced and the host cell-mediated immune response to 16 Minfection was enhanced by inhibitor LY in vitro and in vivo.(3) The apoptosis rates of16M-dependent RAW264.7 macrophages were significantly increased using LY, which wasdependent upon the activities of caspase-3 and caspase-9.(4) The 16M-dependentautophagy was significantly inhibited.(5) After silencing the p110α gene via si RNA, theintracellular survival of 16 M was significantly reduced and the apoptosis rates of16M-dependent cells and cell-mediated immunity were increased and the 16M-dependentautophagy had unchanged. |
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