| Brucella is a facultative intracellular bacterium, which can infect a variety of animals and people, causing significant zoonosis, brucellosis. Not only causing huge losses to the development of animal husbandry, but also seriously threat to human health. Brucella mainly parasitizes both in macrophages and trophoblast cells to complete its life cycle in the host cell,and escaping the host’ immune clearance effection, in order to survive and proliferate in host cells. Research had found that Brucella can inhibit host cells’ apoptosis and ERS has a close relationship with cell apoptosis. In this study, we used B.suis.S2 to infect RAW264.7 and established infection model in vitro to confirm the optimal MOI of cell apoptosis. Using RAW264.7 to establish the infection model and detected the expression of the main factors of ERS, GRP78 and CHOP, in order to analyze the relationship between Brucella and ERS.Meanwhile, we detected the immune factors, IL-1β, IL-6, TNF-α in cell supernatant, to explore the influence of Brucella infection to macrophage’s immunocompetence.1. Using B.suis.S2 to infected RAW264.7 at the MOI of 5∶1, 10∶1, 50∶1 and 100∶1, and collected the cells after 24 h to perform the Hoechst33342/PI assay. The cells started to shown apoptosis when the MOI was 100∶1. Using the MOI of 100∶1 to infect RAW264.7 at0 h, 6h, 12 h, 24 h, 48 h, repectively and collected the cells to perform AO/EB assay. The results shown that RAW264.7 started to apoptosis at 24 h and appeared necrosis at 48 h. Finally, we confirmed the optimal condition is 24 h at a MOI of 100∶1.2. Using RT-qPCR and western blot to detect the expression of GRP78 and CHOP, and we found the expression of GRP78 had a remarkable up-regulation between 0 h and 48 h while the expression of CHOP didn’t change significantly(P > 0.05). The results shown that Brucella infection can induce ERS in RAW264.7, while the apoptosis pathway mediated by ERS didn’t activated. Remind us that Brucella can inhibit or delay the apoptosis of host cells,which is benefit to Brucella’s survive and prolifiration.3. Using RT-qPCR and ELISA to detect IL-1β, IL-6, TNF-α, repectively. The expression of immune factors in infected cells shown significant up-regulation between 0 h and 6 hcompared to the control group(P > 0.05), while the expression of IL-1β and IL-6 appeared a decrease trend with the increase of time. The results shown that Brucella can sitimulated the immunocompetence of macrophage at the initial stage of infection. With the development of infection, the bacterium had a mass proliferation in cells, while its immunocompetence decreased in the process. |
