Effecttion Of Cyclophosphamide On Vivo/Vitro Of Brucella Infection

Object:Brucella is a zoonotic bacterial disease named brucellosis caused by bacteria of the genus Brucella,which are able to establish long-term infections in their hosts.In this paper,the cytotoxicity of cyclophosphamide（CYP）were used to make the tested mice in a temporary state of immunosuppression,so as to explore the process and mechanism of brucella M5infection on immunosuppressed mice.In vitro,cyclophosphamide was used to treat RAW264.7 to investigate the effect of CYP on macrophages’phagocytosis and ability to kill brucellus,as well as the release of related cytokines.It provides a new idea for brucella to induce host inflammatory response and immune escape,and provides data and foundation for further study on the interaction between host cells and pathogens.Methods:Set of immunosuppressant cyclophosphamide（CYP）group,control group（CK）,cyclophosphamide and brucella（CYP+B.melitensis）group and the brucella（B.melitensis）group,each group 40 mg kg^-1 i.p injection of CYP,or amount of normal saline,7 days in a row,after intraperitoneal inoculation 1 x 10⁶ c.f.u brucella,Animals were killed humanely at days 3 and 7and 14.Body weight,food intake,organ mass,coefficient of viscera and body weight,bacteria burden in tissues,immunohistochemical observation,release of IFN-γ,TNF-αand NO cytokines were detected.12.5μmol L^-1,and 25μmol L^-1 of CYP culture RAW264.7 macrophages 2 h,according to the number of macrophages cells infect brucella M5 at propotion of 100:1,cells without CYP named the control group then test intracellular brucella,cells IFN-γ,TNF-αand NO content of cytokines at 2 h,12 h and 24 h,laser confocal microscope cell state,flow cytometry technique to detect 2 h cell to gobble up ability.Results:（1）Mice were injected i.p with 40 mg kg^-1 CYP for 7 days,and loss of weight more than 30%,and the number of peripheral blood white blood cells,lymphocytes and granulocytes decreased more than 50%.（2）The coefficient of organ and body weight of CYP+B.melitensis group and B.melitensis group was significantly higher than that in the CK group（P<0.05）,viscera index of the CYP group is significantly lower than that in the CK group（P<0.05）.（3）CYP+B.melitensis group of spleen,liver and lung had a significantly higher number of living bacterium group of B.melitensis（P<0.05）.（4）The results of immunohistochemical we can draw such a conclusion that CYP increased macrophage infiltration.（5）Serum cytokines IFN-γand NO,CYP+B.melitensis group was significantly higher than that of the CK group（P<0.05）.（6）The 2h after infection,the number of bacterial burden in cells in the CYP group was significantly lower than that the group without CYP（P<0.05）,and was significantly higher at 24h than that in the group without CYP（P<0.05）.（7）The result of flow cytometry we can draw such a conclusion the percentage of fluorecyte in group without CYP was 47.1%,that in the CYP group at 12.5μmol/L concentration was 26.2%,and that in the CYP group at 25μmol/L concentration was 18.1%.Conclusion:kM mice was injected 40 mg kg^-1 CYP i.p for 7 days in a row resulted in temporary and restorable immunodeficiency.In vitro cell experiment results showed that CYP had certain inhibitory effect on RAW264.7 phagocytosis and killing B.melitensis.CYP created a more convenient conditions for brucella in mice,bacterial burden increase in mice,not a consequence of diminished recruitment of immune cells,immunohistochemical results show that increase in macrophage recruitment in mice treated with CYP,and release more cytokines in serum.In conclusion,the increased B.melitensis burden were not a consequence of diminished recruitment of immune cells to the spleen.This phenomena could be explained by decreased cellular microbicidal capacity,the type of cellular infiltration combating the infection or both.