Molecular Mechanism Of Brucella In Inflammatory Signaling Pathways

Brucellosis is bacterial zoonotic infectious disease caused by brucella, infected more than50million each year, especially in developing countries. Brucella spp are the facultative intracellular pathogens with the ability to survive and multiply in phagocytes, and they can cause abortion of pregnant domestic animals and undulant fever in humans. Brucella has unique biological activities, and the complex interaction between the host immune system. At present, the infection of brucella pathogenic mechanism is not enough understanding. Therefore, further clear after brucella infection of the host immune system weakened and its mechanism, for we looking for new types of brucella control strategy provides a new direction and theory basis. Therefore, this study brucella and macrophages in mice as the research object, mainly to carry out the following research work:1. Smooth brucella2308and rough brucella RB51infect RAW264.7macrophages in mice after produce different expression of cytokines. Respectively in2308and RB51brucella infection of mice macrophage, infect4h,8h,24h and48h after using ELISA test kit respectively test the cytokine TNF-alpha, IL-1and IL-10, M-CSF and MIP-2expression quantity;at the same time, With2308brucella and RB51infect macrophages in mice, infection in4h,24h,48h and72h after for brucella Colony-Forming Units. Results found: smooth brucella2308than the rough brucella RB51into the macrophages number, show only can smooth brucella2308in replication intracellular. Compared with smooth brucella2308, rough brucella RB51enter macrophage can produce more inflammatory cytokines, including cytokine IL-10, M-CSF and MIP-2difference was not significant (p>0.05), TNF-alpha and the IL-1difference significant (p<0.05)2. Smooth brucella2308and rough brucella RB51whether activation of MAPK signal pathway and the activation of MAPK signaling pathways affects the expression levels of inflammatory cytokines. With smooth brucella2308and rough brucella RB51infect macrophages after18h for Western Blot detection. Detect brucella2308and RB51infect macrophages in different infection time and different multiplicity of infection the impact of activation of MAPK signal pathway. At the same time with trypan blue staining test whether MAPK signal pathway inhibitors has cytotoxicity. Experimental results show that the rough brucella RB51to activation of MAPK signal pathway, smooth brucella2308is weak and even no activation of MAPK signaling pathways. Rough brucella RB51infect macrophages after eight hours the strongest effect on MAPK signal pathway activity, but the activity of MAPK signal pathway is not as the growth of the brucella infection time increased and when the multiplicity of infection of40, MAPK signal pathways the strongest activity. MAPK signaling pathway inhibitors have no cytotoxicity. First brucella2308and RB51infect macrophages after incubation with a signaling pathway inhibitor, and then detecting cytokines TNF-alpha and IL-1expression level.At the same time, detection signal pathway inhibitors influence for intracellular survival. Results show that the ERK and JNK signaling pathway inhibitor has the concentration dependence and involved in the produce TNF-alpha and IL-1are ERK and JNK signaling pathway, rather than the p38signaling pathway. Compared with control group, the MAPK signaling pathways have influence on brucella intracellular survival.3. The relationship between brucella LPS and MAPK signal pathway.using of purification of brucella LPS and brucella LPS-O chain lack strains infect macrophages respectively, then to find relationship between LPS and MAPK signal pathway. Establishment brucella infection model and extraction of brucella LPS in the first place, then detection with LPS and LPS-O chain deletion mutant infect cells after the activity of MAPK signal pathway. The results show that different concentrations of brucella LPS infect macrophages there was no significant difference to P38, ERK and JNK signaling pathway. With brucella M5-90, the M5-90A WboA and M5-90A pgm of LPS infect macrophages after, no activation of ERK1/2, P38and JNK signaling pathway. Above research show that smooth brucella2308and rough brucella RB51infect macrophages that rough brucella RB51stimulate macrophages can produce more inflammatory cytokines, among them TNF-alpha and IL-1the expression difference significantly. Brucella infected macrophages and found that the rough attenuated strain RB51can activate the MAPK signaling pathway. And to participate in the inflammatory cytokine TNF-alpha and IL-1is a branch of ERK and JNK signaling pathway. Finally, we also found that the Relationship between purification of brucella LPS and MAPK signal pathway was not significant. Through this study, provide clues to reveal the brucella infection of host cells of the host immune system and its mechanism. Also we are looking for new direction and lay the theoretical foundation for brucella control strategy.