| Objectives To investigate how Legionella pneumophila flagellum protein(flaA)and recombinant macrophage inflammatory proteins(mip)would affect the secretion of chemokines and inflammatory cytokines in mouse RAW264.7 macrophages.Methods 1.Grouping: RAW264.7 cells were cultured by flaA(0.00,0.125,0.250,0.500,1.000,2.000,4.000,8.000 μg/mL)or mip(0,0.067,0.136,0.271,0.542,1.084,2.168,4.335 ug/m L).Then EC50 of flaA and mip was determined by CCK-8.The EC50 was diluted by1 /5,1/10 and 1/20 folds: a low-dose flaA group(0.04 ug/mL),a medium-dose flaA group(0.08 ug/mL),a high-dose flaA group(0.16 ug/m L),a low-dose mip group(0.02 ug/m L),a medium-dose mip group(0.04 ug/m L)and a high-dose mip group(0.08 ug/mL),as well as control groups.2.Cell activity detection: RAW264.7 cells were cultured by the above doses of flaA or mip for 24,48 or 72 h.Then cell activity in each group was determined by CCK8.3.RAW264.7 cells were cultured by different doses of flaA or mip for 24 h,and their chemotactic functions were then detected.The supernatants were collected and the secretion levels of chemokines MCP-1 and MIF were detected by enzyme-linked immunosorbent assay(ELISA).4.RAW264.7 cells were cultured by different doses of flaA or mip for 6,12,24,36 or 48 h.Then mRNA expressions of MCP-1 and MIF were detected by quantitative real-time polymerase chain reaction(qRT-PCR).5.RAW264.7 cells were cultured by different doses of flaA or mip for 24,48 or 72 h and their phagocytic functions were then detected.6.RAW264.7 cells were cultured by different doses of flaA or mip for 24,36 or 48 h and the secretion levels of IL-6,IL-1β,IL-1α and IL-10 were detected by ELISA.7.RAW264.7 cells were cultured by different doses of flaA or mip for 6,12,24,36 or 48 h.The mRNA expressions of IL-6,IL-1β,IL-1α,IL-10,IFN-γ,TNF-a,NOD1,NOD2,RIP2,NLRP3 and CASP-1 were measured by qRT-PCR.The protein expressions of NOD1,NOD2,RIP2,NLRP3 and CASP1 were measured by Western blot.Statistical analysis The GraphPad Prism5.0 calculation of recombinant flaA and mip recombinant protein with EC50 software;the measurement data were expressed,analysis of several groups of different time data analysis of variance,using SPSS19.0 statistical software,P < 0.05,the difference was statistically significant.Results 1.Cell activity: The activity of RAW264.7 cells was reduced with the rising dose of flaA or mip.There was a significant difference(P<0.01),with the incubation time to extend the activity decline trend(P<0.01),24 h was better than other time periods,with 72 h decreased significantly,different time interaction effect(P<0.05).The dose for halfmaximal50% inhibitory concentration(EC50)of flaA was determined on Data were statistically analyzed on GraphPad Prism 5.0.indicating the optimal cultivation period was 24 h.The EC50 of flaA was 0.8 μg/m L,which was significantly the best for the activity of RAW264.7 cells.indicating the optimal cultivation period was 24 h.The EC50 of mip was 0.43 ug/m L,which was significantly the best for the cells.2.The flaA and mip both promoted the secretion of chemokines in RAW264.7 cells.After cultivation by different doses of flaA or mip by 24 h,The secretion of MCP-1 and MIF was promoted by the increasing doses of recombinant proteins,especially at the doses of high doseincreased significantly,which were significantly different from the control groups and other test groups.3.The cultivation with flaA or mip promoted the mRNA expressions of MCP-1 and MIF in RAW264.7 cells.After cultivation with different doses of flaA or mip for 6,12,24,36 or 48 h,the secretions of MCP-1 and MIF by RAW264.7 cells were both promoted.The secretions were maximized after 12 h of cultivation with high dose increased significantly,but were reduced with the prolonging of time,which were significantly different from the control groups and other test groups.4.After cultivation by flaA or mip,the phagocytic functions of RAW264.7 cells were reduced.After culture with different doses of flaA or mip for 24,48 or 72 h,the phagocytic functions of RAW264.7 cells were weakened with the rising doses of recombinant proteins and with the prolonging of time.5.After cultivation with different doses of fla A or mip for 24,36 or 48 h,the secretions of IL-6,IL-1β,IL-1α and IL-10 by RAW264.7 cells were all induced,though to different degrees.The secretion was enhanced with the rising dose of recombinant proteins.The secretions were maximized after 36 h of cultivation with high dose increased significantly,but were reduced with the prolonging of time,which were significantly different from the control groups.6.The cultivation with flaA or mip promoted the mRNA expressions of IL-6,IL-1β,IL-1α,IL-10,IFN-γ,TNF-α,NOD1,NOD2,RIP2,NLRP3 and CASP-1 in RAW264.7 cells.The secretions were maximized after 12 h of cultivation with high dose increased significantly,but were reduced with the prolonging of time,which were significantly different from the control groups and other test groups.7.After cultivation with different doses of fla A or mip for 6,12,24 or 48 h,the secretions of NOD1,NOD2,RIP2,NLRP3 and CASP-1 by RAW264.7 cells were all promoted.The secretions were maximized after 24 h of cultivation with high dose increased significantly,but were reduced with the prolonging of time,which were significantly different from the control groups and other test groups.Conclusions 1.The culture with recombinant flaA or mip increasech macrophages chemotactic function and cytokine secretion.Downregulation of phagocytosis.2.The recombinant flaA could promote the secretion of cytokines in cells by stimulating the NLRP3 and CASP-1.3.The recombinant mip could promote the secretion of cytokines by stimulating the NOD1,NOD2 and RIP2. |
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