| Bovine Pasteurella multocida is an important pathogen of cattle can cause hemorrhagic septicemia and pneumonia diseases which bring huge economic losses. P. multocida was divided into 5 serotypes including A, B, D, E and F serotype based on capsular antigen. Serotype B and E main cause bovine hemorrhagic septicemia, serotype A and F main cause bovine respiratory disease leading pneumonia. In recent years, with the rapidly development of cattle-breeding industry, the transport stress caused by long-distance and frequent transportation made the increasing of pasteurellosis in cattle. At present, the pathogenesis of bovine P.multocida is poorly understood, and the research in this area is also very few. Upon to the study of inflammatory signaling pathways of mammal cells induced by bovine P.multocida, here we want to illuminate the pathogenic mechanism of P.multocida to provide some ideas for new control strategies and new vaccines development in the control of bovine Pasteurellosis. In this experiment, mouse macrophage RAW264.7 cells were chosen as target cell to investigate the cell inflammatory reaction induced by bovine P.multocida setotype A(Pm A) or serotype B(Pm B) detected by q RT-PCR, Western Blot, ELISA and other techniques. The main objectives of this research were as follows: to investigate whether(1) the inflammatory response of macrophages induced by Pm B and Pm A in vitro conditions;(2)Toll-like receptors were involved in activation of inflammatory signaling pathway;(3)NF-κB, MAPK signaling pathway were involved in the regulation of inflammatory signaling pathways;(4) inflamsomes were involved in the regulation of inflammatory inflammatory signaling pathway;(5)phagocytosis were impacted on the modulation of inflammatory signaling pathway.The results are as follows:1. Inflammatory response of macrophage cells induced by Pm A and Pm B Macrophage RAW264.7 were dealt with different multiplicity of infection(MOI) of Pm A and Pm B, respectively, and the macrophage cells and supernatants were collected at 0.5 h, 1 h, 2 h, 3 h post infected. The expressions of IL-1β, IL-18 were detected using q RT-PCR and Western Blot, and the secretion of inflammatory cytokines IL-1β, IL-18 and TNF-α’s were detected by ELISA. The results showed that the IL-1β m RNA of macrophages was increased along with dose-dependents challenged with Pm A and Pm B, respectively; protein expressions of IL-1β, IL-18 and the secretion of IL-1β, IL-18, TNF- α were also significantly increased, which indicated that Pm A and Pm B can activate the inflammatory reaction of macrophage cells.2. Inflammatory signaling pathways mediated by TLR2 and TLR4 In order to investigate that whether the Toll-like receptor signaling pathways can be involved in activation of inflammatory, the macrophage cells were treated with 10 MOI of Pm A and Pm B for 2 h, respectively, and then the m RNA expression levels of TLR1,2,3,4,5,6,7,8,9 were detected using q RT-PCR. The results showed that m RNA expressions of TLR2, TLR4 were significantly increased but not TLR1,3,5,6,7,8,9, which indicated that TLR2, TLR4 were involved in the regulation of inflammatory signaling pathways induced by Pm A and Pm B, respectively. 3. NF-κB, MAPK involved in the regulation of inflammatory signaling pathways3. NF-κB, MAPK involved in the regulation of inflammatory signaling pathways To access the inflammatory regulatory roles of P38, JNK, NF-κB signaling pathway in macrophage infected by Pm A and Pm B,respectively, here the macrophages were treated with 10 μmol of P38, JNK, NF-κB signaling pathway inhibitors(SB203580, SP600125, PDTC) for 1 h first, and then dealt with 10 MOI of Pm A, Pm B. The m RNA level of IL-1β was detected using fluorescence quantitative q RT-PCR. The results showed that the expressions of IL-1β of macrophages dealt with P38 or NF-κB signaling pathway inhibitor first and infected Pm A later were significantly decreased, and the expressions of IL-1β of macrophages dealt with JNK, P38 or NF-κB signaling pathway inhibitor first and infected Pm B later were also significantly decreased, which indicated that P38, NF-κB were participated in inflammatory reaction induced by Pm A and JNK, P38, NF-κB were involved in inflammatory reaction induced by Pm B. 4. Inflamsomes involved in the activation of inflammatory Signaling pathways In order to investigate the role of inflamsomes in inflammatory signaling pathways,4. Inflamsomes involved in the activation of inflammatory Signaling pathways In order to investigate the role of inflamsomes in inflammatory signaling pathways,the macrophages infected with 10 MOI of Pm A, Pm B were collected at different time points to detect the m RNA expression levels of NLRP1, AIM2, NLRP3, NLRC4, NLRP6 and inflamsome joint protein ASC using fluorescence quantitative q RT-PCR and to detect the protein level of NLRP3 using Western Blot. Collected cells dealt with 0.8μmol Caspase-1 inhibitor VX-765 to detect the m RNA expression level of IL-1β by q RT-PCR. The results showed that the expressions of NLRP3 and NLRP6 were significantly increased but not of NLRP1, AIM2, NLRC4, which indicated that NLRP3, NLRP6 were involved in regulation of the inflammatory signaling pathways induced by Pm A and Pm B, respectively. The m RNA expression level of ASC was also significantly increased and the increasing trend was consistent with IL-1β m RNA expression. The activation of inflammatory composite adapter protein ASC indicated that inflamsomes were involved in the inflammatory signaling pathways of Pm A, Pm B induced. The m RNA expression of IL-1βwas significantly inhibited by VX-765 inhibitor which indicated that the expression of IL-1β was dependent on Caspase-1, and its important role in activation of inflammatory induced by Pm A and Pm B, respectively, and it also can be used as a indicator of activation of the inflammatory. 5. NF-κB, MAPK signaling pathways involved in the regulation of inflamsomes5. NF-κB, MAPK signaling pathways involved in the regulation of inflamsomes To illuminate the relationships between P38,JNK, NF-κB signaling pathwaysand inflamsomes, the macrophages dealt with 10 μmol of P38, JNK, NF-κB signaling pathway inhibitor SB203580, SP600125, PDTC for 1 h were treated with 10 MOI of Pm A and Pm B,respectively, the cells were collected and then the expressions of NLRP3, NLRP6 were detected by fluorescence quantitative q RT-PCR and Western Blot,. The results showed that the expressions of NLRP3 and NLRP6 of macrophages dealt with inhibitors were significantly decreased and increased, respectively, which indicated that P38 and NF-κB signaling pathways were the positive regulation to NLRP3 and negative regulation to NLRP6, JNK was no regulation to NLRP3 but had significantly negative regulation to NLRP6. 6. Phagocytosis involved in activation of inflammatory signaling pathways6. Phagocytosis involved in activation of inflammatory signaling pathways In order to investigate the relationship between phagocytosis and, inflammatory signaling pathways induced by Pm A, Pm B, the cells were dealt with cytochalasin D for 1 h which made the cells lost its phagocytosis and then treated with 10 MOI of Pm A and Pm B for 2 h, respectively. The m RNA expressions levels of IL-1β, ASC, NLRP3, NLRP6 were detected using q RT-PCR. The results showed that the expressions of IL-1β, ASC and NLRP3 were significantly decreased, but NLRP6 significantly wereup-regulated, which indicated that phagocytosis was involved in the inflammatory signaling pathways induced by Pm A and Pm B, respectively.7. Conclusion(1) The activation of inflammatory signaling pathways of murine macrophage can be induced by Pm A, Pm B, and TLR2, TLR4 were involved in inflammatory.(2) P38, NF-κB signaling pathway were involved in the inflammatory signaling pathways induced by Pm A, and JNK, P38, NF-κB signaling pathway were involved in the inflammatory signaling pathways induced by Pm B.(3) Inflamsome NLRP3 and NLRP6 were involved in the inflammatory signaling pathways induced by Pm A and Pm B, respectively.(4) JNK, P38, NF-κB signaling pathway were positive regulation to NLRP3 and negative regulation to NLRP6 among the inflammatory signaling pathways induced by Pm A and Pm B, respectively.(5) Phagocytosis was involved in the activation of inflammatory signaling pathways induced by Pm A and Pm B, respectively. |
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