Research On The Regulation Mechanism Of Brucella Infection On NF-kappa B Signaling Pathway

Macrophages are the main target cells for the survival and reproduction of Brucella.Brucella can affect the macrophage apoptosis pathway through certain apoptosis-related factors,preventing itself from being phagocytosed by macrophages,so that it could be able to reproduct persistently in host cells,thus the intricate interaction molecular mechanism between Brucella and host cells is also one of the reason for the long-lasting brucellosis.Research had shown that NF-?B is involved in many cellular activities such as inflammation,apoptosis,immune response and cell cycle regulation et al.But the mechanism of action of the protein in Brucella-induced apoptosis and inflammation is not yet clear.This research established a theoretical basis for revealing the pathogenic mechanism of Brucella infection by studying the apoptosis and inflammatory response which is induced by Brucella and the relationship between NF-?B and its downstream target genes.Objective:In this research,the mouse macrophage RAW264.7 model infected by Brucella bovis 2308 strain,which was used to screen downstream regulatory genes of the NF-?B transcription factor,so as to research the function of the gene sequence in the process of Brucella infection.It reveals the effect of Brucella infection on inflammation and apoptosis regulated by the NF-?B signaling pathway in mouse mononuclear macrophages.So as to provide a new idea for further research on the mechanism of Brucella infection.Methods:The siRNA was designed to interfere with Brucella infected RAW264.7 cells,and the relative expression levels of NF-?B P65 mRNA was detected by RT-PCR.RAW264.7 cells infected with Brucella 2308 were treated with NF-?B inhibitors,NLRP3inhibitors,TGF-?inhibitors,RT-PCR methods were used to verify interactions relationship between the two others in RAW 264.7 cells infected by the Brucella 2308.The CHIP test was performed between the Brucella infected RAW264.7 cells group and non-infected cells group.High-throughput sequencing technique was used to screen out differentially-targeted genes regulated by NF-?B.Furthermore,P65 siRNA and target genes Interference test,through the fluorescence quantitative analysis and flow cytometry techonology detecting whether RAW264.7 cells infected by Brucella bovis 2308 affected the apoptosis and inflammatory response,so that it confirmed whether the RAW264.7 cells is regulated by NF-?B gene that have an effect on apoptosis and inflammation.Results:?1?Compared with normal RAW264.7 cells,RAW264.7 infected by Brucella2308 could significantly increase the relative expression level of NF-?B,while the interference of P65-siRNA and P65-shRNA could inhibit the expression of NF-?B P65protein relative expression level.Compared with the control group?DMSO group?,in NF-?B inhibitor groupthe expression of TGF-?₁,IL-1?and IL-18 was significantly higher than that in DMSO group.The expression of TGF-?₁ was significantly higher than that in negative control group in NLRP3 inhibitor group.The expression of IL-1?and NLRP3 was significantly higher than that in negative control group in TGF-?inhibitor group.The relative expression levels of NF-?B,IL-1?,IL-18 and NLRP3 up-regulated,and the relative expression of TGF-?₁ down-regulated in the Brucella infection group.The concentration of IL-18 was gradually decreased from 0 to 24 h except for 12 h in the TGF-?inhibitor group.In the NLRP3 inhibitor group,the TGF-?factor concentration gradually increased at 4-12 h except 0h and 24 h.In addition to 0 h,the IL-1?factor concentrations of 4-24 h gradually decreased.In the NF-?B inhibitor group,TGF-?factor gradually decreased from 0 to 24 h except for 4 h.Except for 24 h,the concentrations of IL-18 decreased gradually from 0 to 12 h.?2?Detected by the CHIP-seq technique,411 different genes were obtained from the RAW264.7 cell infection group,and 539 different genes were obtained from the PBS negative control group.The research screened the peak located in the promoter region near the transcription initiation site where is the downstream of the NF-?B P65 target genes:9 different genes in infection group and 7 different genes in negative control group.In this research,we screened three genes that might be associated with apoptosis and inflammation in infection group for subsequent studies:Stap2,Igf1R,and Mir682.Inhibition of Brucella-infected RAW264.7cells by NF-?B inhibitors,Brucella infected for 24 h,RT-PCR results showed that the NF-?B inhibitor group significantly increased the expression of Stap2 relative to the DMSO group.The expression levels of Stap2 and Igf1R in the infection group were significantly increased.?3?The results of RT-PCR and flow cytometry showed that Igf1R siRNA significantly increased the expression of ASC?P<0.05?and gradually increased at 4-24 h.The expression of Caspase-3 under the irradiation of Mir682 inhibitor was gradually increased,and it was significantly expressed at 24 h?P<0.01?.Except for 8 h,Stap2 siRNA gradually decreased ASC expression and it was significantly expressed at 4 h.The expression of AIM-2 increased gradually at 4²4 h after Brucella infected RAW264.7 cells.The apoptosis rate of interfering gene cells was detected by flow cytometry at 12 and 24 h after infection with Brucella 2308:the apoptosis rate of Stap2 siRNA group,Mir682 inhibitor group,and Igf1R siRNA group at12 h was lower than that of NC group at 24 h.Afterwards,the apoptotic rates of the Stap2siRNA group,the Mir682 inhibitor group,and the Igf1R siRNA group were higher than that of the NC group.The apoptosis rate of Stap2 siRNA group at 24 h was significantly different from that of NC group?P<0.01?.In the Infection group of Brucella 2308,the rate of cell apoptosis showed a slight decrease from 12 to 24 hours.Conclusion:?1?Compared with the normal RAW264.7,RAW264.7 infected by Brucella2308 promotes the expression of NF-?B and NLRP3 and NF-?B promotes the expression of NLRP3 and TGF-?₁.NLRP3 promoted the expression of TGF-?₁ and NF-?B.TGF-?promoted the expression of NLRP3.?2?Compared with the non-infected group,the expression of NF-?B was up-regulated after Brucella infection of RAW264.7 cells,which promoted the expression of Stap2 and Igf1R.Igf1R,Stap2,and Mir682 inhibited apoptosis and inflammation after Brucella infecting the RAW264.7 cells.