The Preliminary Study On The Regulation Of RAW264.7 Macrophage Function By Recombinant Protein IP,MOMP And PILE Of Legionella Pneumophila

Objective To analy the functions of the recombinant protein IP,MOMP and PILE about the RAW264.7 macrophages,such as its phagocytosis,migration function,inflammatory factors secretion,explore the mechanism of its functions.Methods The RAW264.7 macrophages were co-incubated with the target protein who was purified by IDA His · Bind resin in vitro.CCK-8 Kit was used to detect the virulence of the target protein to RAW264.7 macrophages and the IC50 of the cells was counted.The experiments were divided into four groups: IC50 / 5,IC50 / 10,IC50 / 20 as the experimental group,RAW264.7 macrophages with culture medium were used as the control.The phagocytosis of RAW264.7 macrophages was detected by neutral red phagocytosis for 24 h,48 h and 72 h respectively.After 24 h incubation,the migration function of RAW264.7macrophages was detected by Transwell chamber,the levels of chemokines MCP-1 and MIF in the supernatant were also measured.The levels of IL-1α,IL-1β,IL-6 and IL-10 in the culture supernatant were detected by ELISA kit for 24 h,36 h and 48 h.After incubation for 6h,12 h,24 h,36 h and 48 h,the levels of IL-1α,IL-1β,IL-6,IL-10,MCP-1,MIF,TNF-α,IFN-γ were detected by real-time quantitative PCR from the mRNA expression levels.The expression levels of NOD1,NOD2,RIP2,NLRP3 and caspase-1 were detected by q-PCR and western-blot respectively from mRNA level and protein level.The statistical data were processed by SPSS19.0 software.The results were expressed as means ± standard deviation(SD).For single-factor analysis of variance,the K-independent samples were used for non-normal distribution.Nonparametric test,P <0.05 for the differencewas statistically significant.Graph Drawing was performed using GraphPad Prism 5.0software.Results 1.The recombinant protein IP(46 kDa),recombinant protein MOMP(50 kDa),and recombinant protein PILE(35.7 kDa)were induced and purified by IPTG.2 The cck-8results were that the half of the lethal concentration about recombinant protein IP,MOMP,PILE were 6.24μg / mL,5.69μg / mL,6.00μg / mL respectively.3.Compared with the control,as the concentration and the time about target protein IP,MOMP and PILE increased,the ability to devour the neutral red about RAW264.7 macrophages were decreased(P <0.05).4.Compared with the control group,as the concentration about target protein IP,MOMP and PILE increased,the migration function of RAW264.7 macrophages was also increased(P<0.05).5.The secretion of MCP-1 and MIF in the culture supernatant of Transwell cells was significantly higher than that of the control group(P <0.05).As the concentration about recombinant protein increased,its secretion increased(P <0.05).6.The levels of IL-1α,IL-1β,IL-6 and IL-10 in the experimental group were significantly higher than those in the control about the RAW264.7 macrophages treated with IP,MOMP and PILE(P <0.05),the inflammatory factors increased with the protein concentration(P <0.05),and its secretion reached the peak at 36 h,and then began to decrease.7.Compared with the control,the mRNA expression levels of IL-1α,IL-1β,IL-6,IL-10,TNF-α,IFN-γ and chemokine MCP-1,MIF were increased(P <0.05).8.Compared with the control group,after 6h,12 h,24h,36 h and 48 h co-culture with macrophages,the mRNA expression of NOD1,NOD2,RIP2,NLRP3 and caspase-1 in the IP and PILE groups were significantly increased,and reached the peak at 12 h(P <0.05);The mRNA expression levels of NOD2,RIP2,NLRP3 and caspase-1 were increased in the target protein MOMP group,and reached the peak at 12h(P <0.05).9.Compared with the control group,after co-culture with the macrophages for 12 h,24h and 36 h,the protein expression level of NOD1,NOD2,RIP2,NLRP3 and caspase-1 in the IP and PILE groups were increased,reached the peak at 24h(P <0.05).The protein expression levels ofNOD2,RIP2,NLRP3 and caspase-1 in the target protein MOMP group were increased,and reached the peak at 24 h(P <0.05).Conclusion The recombinant protein IP,MOMP and PILE can down-regulate the phagocytosis and up-regulate the migration of RAW264.7 macrophages;The expression of IL-1α,IL-1β,IL-6,IL-10,TNF-α,IFN-γ,MCP-1 and MIF were up-regulated;The recombinant protein IP and PILE maybe via the NOD1 / NOD2 / RIP2 and NLRP3 / caspase-1signaling pathways to regulate the function of macrophage,but the mRNA and protein expression levels about NOD2 / RIP2 were higher than NOD1 / RIP2.Maybe the NOD2 /RIP2 and NLRP3 / caspase-1 signaling pathways were used to regulate the function of macrophage about MOMP protein.