The Preliminary Study On The Role Of NLRP3-Inflamma-Some During The Infection Of Brucella

The Brucellosis is a worldwide zoonosis,which brings seriously harm to the human health andthe development of animal husbandry. For the lack of safe,effective vaccines and convenient,low-cost means of detection,a breakthrough on the prevention and control of Brucella is sodifficult to be achieved.In addition, with the rapid development of the logistics of live animals andanimal products,the prevalence of Brucella in China has increased year by year.Brucellas,whichhave complex mechanisms that are different to many other Gram-negative bacteria and pathogenic,does not cause a strong inflammatory response in the macrophages at initial infection stage,noractives strong innate immune activation and thus evades the body ’s resistance to them.During theperiod of settlement in macrophages, Brucella inhibit the apoptosis to extend the time for survivaland reproduction in intracellular. Innate immunity is the first line for body to resist the pathogenicbacterium and inflammatory is also an important pathologic response to the invading pathogenicmicroorganisms.One of the way to trigger and active the two pathway is the pattern recognitionreceptors (PRRs),which include an important NLRPs family.One of the member of NLRPs isNLRP3,the constituents of the NLRP3-inflammation which plays an important role in theperception of the damage associated molecular patterns and dangerous (DAMPs)such aspathogenic microorganisms, foreign bodies and some macromolecular substances released by thecell. So research on the relationship between the NLRP3-inflammasome and the Brucella plays animportant role in providing experimental evidence for the mechanism of the innate immune andinflammatory responses for the Brucellosis.Objective:1. Make it clear that the transcription distribution of the pattern recognition receptorsNLRPs in mouse organs and macrophages and select the organs and cell expressing NLRP3as theresearch object.2.Clarify the change of the NLRP3-inflammasome and the related cytokine in bothcell and animal infected by the Brucella.3.Detect the change of the NLRP3-inflammasome incells contacted with the Brucella LPS.4.Reveal the relationship between NLRP3inflammasomeand the intracellular Brulella.Methods: RT-PCR method was used to detect the transcription of the NLRPs genes in mouseheart, liver, kidney, spleen, lung, intestine, placenta in pregnant rats and the mouse macrophageRAW264.7;The cells models and animal models infected by the Brucella were established for thelast research.qRT-PCR was applied to detect the NLRP3, ASC, Caspase1, IL1-β, IL18levels incells infected by Brucella for4h,8h,12h and24h and that in mice livers, spleens and kidneysinfected by Brucella for1week,2weeks,3weeks and4weeks. The secretion of IL1-β and IL18in the supernatant of RAW264.7cells and in the mouse serum were detected through ELISAmethod.The models in which RAW264.7cells were added with different concentrations of Brucella2308LPS, RB51LPS and△WbkA LPS were established for the detection ofNLRP3,ASC, Caspase1,IL1-β and IL18by qRT-PCR.The pathological changes in mouseliver,spleen and kidney infected by Brucella were observed by the method of tissue sectiontechnique; The distribution and expression of the ASC in mouse liver,spleen and kidney infectedby Brucella were displayed by the method of Immunohistochemistry.Results: Many members of the pattern recognition receptors NLRPs genes are transcripted in themouse liver, spleen, kidney, heart,small intestine and placenta, whereas the mouse lung andmacrophages only transcript the NLRP3and NLRP4efg. NLRP3was widely transcripted in themacrophages and all the tissues above.NLRP3-inflammasome related genes was mostly shortlyreduced at the early infection stage in both RAW264.7and THP-1cells infected by theBrucella,and so do the expression of IL1-β and IL18in the supernatant of RAW264.7cells.themost efficient concentration of2308LPS and RB51LPS to active the NLRP3-inflammasomeranges from1ng/ml to10ng/ml.The△WbkA hardly active the NLRP3-inflammasome, but it caneffectively stimulate the transcription of IL1-β and IL18. Brucella2308, RB51,△WbkA andListeria could lead to the activation of the NLRP3inflammasome in the liver, spleen and kidney.Compared with RB51and Listeria,2308can strongly up-regulate the NLRP3-inflammasomeand the related cytokine in mouse liver, spleen and kidneys, but the effect of△WbkA is so weakand even down-regulate some genes in some time point. The change of the theNLRP3-inflammasome and the related cytokine actived by2308, RB51and Listeria in mouseliver, spleen and kidney show a “wavy” trend.Inflammatory pathological changes wereobserved such as lymphocyte infiltration in the liver,epithelial cells nodules in the spleen andtubular epithelial cell swelling in kidney;The expression of ASC in mouse liver,spleen and kidneyshowed a role of2303infected group>RB51infected group>△WbkA infected group.