The Molecular Sterilization Mechanisms Research Of Leptospira Affect By Different Host Macrophages

Leptospies disease is an epidemic Amphixenosis caused by Leptospira interrogans which found all over the world,pathogenic Leptospies invades the human body due to acute fever and other systemic diseases.One of the main hosts of leptospirosis is rodents,but the mice does not infected by leptospirosis.From recent report,Leptospies interrogans were killed in murine macrophages,while living and reproduction in the memory of human macrophages.The mechanism is not clear.At the early stage of leptospirosis infection,reactive oxygen intermediates(ROIs)and activity of nitrogen intermediates(RNIs)pathway is classical pathway for killer cell inhibitory bacteria.In this research,the mechanism and bacteria inhibitory pathway on different host macrophages phagocytosis L.interrogans bactericidal were studied,the L.interrogans serovar Lai strain infected human or mouse mononuclear macrophage model were built,the founction and the difference of L.interrogans ROIs,RNIs pathway were exposited.The summarization of molecular sterilization mechanisms of Leptospira affect by different host macrophages were discussed.1 ROIS system of Leptospies interrogans in human and mouse macrophage apoptosis memory:(1)Detection of NADPH oxidase activity in macrophages:By Spectrophotometry with prototype nicotinamide adenine dinucleotide phosphate(NADPH)blocking experimental detection of L.interrogans uninfected and infected 2h,4h,12h and 24h mouse mononuclear-macrophage like cell line(J774A.1)and human mononuclear cells(THP-1)and the activity of NADPH oxidase.Results show activity of NADPH oxidase in J774A.1cells by infection before 0.6190?mol/min/mg raised 2.48,9.92,5.16 and 4.29 times(P<0.05),NADPH blockers,the activity is not blocked group of 0.20,0.41,0.09 and 0.21times,blocking agent treatment,enzyme activity decreased significantly(P<0.05);After infected by L.interrogans THP-1 cells 2h,4h,12h and 24h,NADPH oxidase activity is infection 0.7235?mol/min/mg 1.22,6.09,2.06 and 1.33 times(P<0.05);after blockade of NADPH.Activity is not blocked group of 0.45,0.38,0.76 and 0.58 times(P<0.05);(2)Detection of ROS levels in macrophages:The ROS specific fluorescent probe DCFH-DA markers,with NADPH examined by fluorescence microscope before and after the blockade of L.interrogans uninfected and infected during different periods of J774A.1and THP-1 cells and ROS level.The experimental results show that the infection,2h?4 h?12 h and 24 h after J774A.1 ROS specific fluorescence intensity compared with infection obviously enhanced,after blockade of NADPH fluorescence brightness significantly weaker,blocking effect is obvious,and THP-1 cells after infection the ROS fluorescence brightness changes and blocking effects are not obvious;(3)The content of O₂-in the supernatant of macrophage cell:By using nitroblue tetrazolium(NBT)reduction method of commercial kits NADPH before and after the blockade of L.interrogans uninfected and infected with different time periods in J774A.1and THP-1 cells in O₂-level.The results showed that the L.interrogans infection J774A.1cells 2h,4h,12h and 24h after O₂-detection OD_450nmvalues by pre infection 0.1890 raised to 1.88,1.79,1.90 and 1.79 times(P<0.05)and NADPH was blocked after the results are not blocked group of 0.67 0.88 and 0.90 and 0.90 times(P<0.05);THP-1 cells infected with Leptospies detected the OD value of O₂-by pre infection 0.1237 raised to 205,216,2.19 and 2.48 times(P<0.05),NADPH was blocked after the result is not blocked group of 0.59,0.93,1.00 and 0.89 times(P<0.05).(4)The intracellular phagosome,body shape,number and distribution of observation:By transmission electron microscopy(TEM)NADPH before and after blocking L.interrogans infection in different time periods in J774A.1 and THP-1 cells phagocytic vacuole membrane,Leptospies morphology,number and distribution.Observation results show that L.interrogans strain 56601 in J774A.1 cells were swallowed bubble surrounded by a membrane,with the extension of the infection time,intracellular Leptospies decline and typical pattern of Leptospies interrogans become incomplete,but after blocker treatment by Leptospies in J774A.1 cells were swallowed bubble surrounded by a membrane decreased significantly,with the extension of the infection time,cell number of Leptospies interrogans increased,and Leptospies interrogans Leptospies maintain complete typical morphology.And the majority of Leptospies interrogans in blocking agent treatment of THP-1 cells were free in the cytoplasm of a cell,intracellular Leptospies interrogans counts were increased with the extension of the infection time of Leptospies were maintained complete typical morphology.(5)To observe the co-localization of intracellular Leptospies and lysosomal LAMP-1:Fluorescent antibody staining after laser scanning confocal microscope,NADPH before and after the blockade of L.interrogans infection during different periods of J774A.1 and THP-1 Cell Leptospies and lysosome associated membrane protein 1(LAMP-1)Co localize with.Observations reveal,J774A.1 cells in Leptospies and LAMP-1co-localization increased with the extension of the infection time(P<0.05),intracellular Leptospies specific fluorescence gradually changed little,but the blocking agent blocking agent in the treatment of Leptospies and LAMP-1 co-localization with the prolongation of the time of infection and decreased significantly(P<0.05).LAMP-1 co-localization gradually reduced with time of infection time of THP-1 Cell Leptospies and lysosomal membrane logo,intracellular Leptospies specific fluorescence gradually increased(P<0.05),the blocker of Leptospies and THP-1 Cell lysosomal membrane LAMP-1co-localization had no significant effect.(6)In macrophages Leptospiesl viability assay:By flow cytometry NADPH before and after the blockade of L.interrogans infected cells intracellular Leptospies interrogans activity.The results showed that the J774A.1 from before infection of 97.03%,down to pre infection 0.942,0.851,0.756 and 0.724 times(P<0.05)and NADPH was blocked for non blocking group at the same time paragraph 1.17,1.67,1.93 and 2.36 times(P<0.05).THP-1 Cell Leptospies activity from before infection of 96.87%,reduced to pre infection0.898,0.894,0.826 and 0.864 times(P<0.05)after blockade of NADPH by is not blocked group of 0.89,1.00,1.02 and 0.98 times(P<0.05).2 RNIS system of Leptospies interrogans in human and mouse macrophage apoptosis memory(1)Macrophage i NOS m RNA levels were detected:GAPDH for reference,by real-time fluorescent quantitative real time PCR detection of L.interrogans uninfected and infected during different periods of J774A.1 and THP-1 Cell i NOS m RNA levels.The results showed that the strains infecting J774A.1 cells 2h,4h,12h and 24h Lai and i NOS m RNA expression level compared with uninfected cells were raised 1.37,2.82,25.76 and 27.47times(P<0.05)and Leptospies interrogans serovar Lai strain infected THP-1 Cell i NOS m RNA expression level compared with uninfected cells up-regulated 1.59,3.98,3.89 and8.81 times(P<0.05).(2)Mmacrophage i NOS protein levels were detected:combining immunofluorescence labeling of i NOS protein immunofluorescence staining,by flow cytometry mark i NOS blockade and uninfected and infected during different periods of J774A.1 and THP-1 Cell i NOS protein levels.The results show that Leptospies infection J774A.1 cell i NOS protein expression rate by the infection of 34.16%rise to 8.94,9.77,9.56 and 9.76 times(P<0.05),i NOS blocking agent blocking is not blocked group of 0.82,0.64,0.57 and 0.52times(P<0.05).The expression rate of i NOS protein in THP-1 cells increased from22.08%to 2.13,2.38,2.35 and 1.92 times(P<0.05)before infection,and i NOS was 0.93,0.86,1.11 and 1.03 times of the non blocking group(P<0.05).(3)Macrophage NO level detection:using Griess assay for the detection of Leptospies interrogans i NOS blocking agent before and after the blockade of uninfected and infected at different time in J774A.1 and THP-1 cells in the supernatant of NO content.The results showed that L.interrogans infection J774A.1 cells of NO expression from before infection0.1588 mol/L rose to 6.75,644,6.99 and 6.51 times(P<0.05);after inhibition of i NOS is not blocked group of 0.50,0.49,0.64 and 0.73 times(P<0.05);THP-1 cells after infection with no expression by infection of 0.0988 mol/L rose to 2.88,3.27,2.80 and3.35 times(P<0.05).After blocking is not blocked group of 1.00,0.93 and 0.94 and 1.00times(P>0.05).(4)Within the macrophage phagocytic vacuole,Leptospies morphology,number and distribution of observation:By transmission electron microscopy to detect the expression of i NOS blockade blockade and Leptospies interrogans infection in different time periods in J774A.1 and THP-1 cells phagocytic vacuole membrane,Leptospies morphology,number and distribution.Observation results show that L.interrogans strain 56601 in J774A.1 cells were swallowed bubble surrounded by a membrane,with the extension of the infection time,intracellular Leptospies decline and typical pattern of Leptospies interrogans become incomplete,but after blocker treatment by Leptospies in J774A.1 cells were swallowed bubble surrounded by a membrane decreased significantly,with extension of the infection time,cell number of Leptospies increased,Leptospies and maintain complete typical morphology.And most of the Leptospies in blocking agent treatment of THP-1 cells were free in the cytoplasm of a cell,intracellular Leptospies counts were increased with the extension of the infection time of Leptospies were maintained complete typical morphology.(5)In macrophages in Leptospies and lysosomal LAMP-1 co localization observation:combined with fluorescent antibody staining,using laser scanning confocal microscope to detect the expression of i NOS blocking blocking agents in different time periods in J774A.1 and THP-1 Cell Leptospies interrogans and lysosome LAMP-1 co-localization of the situation before and after the Leptospies interrogans infected.Observations reveal,J774A.1 cells in Leptospies and LAMP-1 colocalization increased with the extension of the infection time(P<0.05),intracellular Leptospies specific fluorescence gradually changed little,but the blocking agent blocking agent in the treatment of Leptospies and LAMP-1 co-localization with the prolongation of the time of infection and decreased significantly(P<0.05).LAMP-1 colocalization with infection from time to time prolonged(P<0.05)decrease of THP-1 Cell Leptospies and lysosomal membrane logo,intracellular Leptospies specific fluorescence gradually increased,the blocker of Leptospies and THP-1Cell lysosomal membrane LAMP-1 co-localization has no obvious influence.(6)In macrophages Leptospiesl viability assay:using flow cytometry to detect the expression of i NOS blocking agent blocking before and after infected by L.interrogans cell intracellular Leptospies activity.The results showed that J774A.1 was down to 0.942,0.756,0.851 and 0.724 times(P<0.05)before infection,1.18,1.58,1.89,97.03%and 1.77times of the non blocking group(P<0.05).THP-1 Cell Leptospies activity from before infection of 96.87%,reduced to pre infection 0.898,0.894,0.826 and 0.864 times(P<0.05)after inhibition of i NOS by,the result is not blocked group of 1.01,0.99 and 1.04 and 1.01times(P<0.05).Conclusion:1.Infected by L.interrogans J774A.1 and THP-1 cells NADPH oxidase activity,ROIs of superoxide ion levels and ROS levels were significantly up-regulated,but raised by the infection of mouse macrophages is more significant.ROIs pathway blockade results show,in mouse macrophage of Leptospies were swallowed bubble surrounded by a membrane decreased with time prolonged infection,Leptospies morphology is clear and the number gradually increased,intracellular Leptospies and lysosomal membrane total decreased gradually,whereas the co localization of THP-1 cells within lysosomes and Leptospies was slightly reduced,a slight increase in the number of Leptospies,ROIs tips are involved in bactericidal effect of mouse and human macrophages of Leptospies interrogans.2.Infected by L.interrogans after J774A.1 and THP-1 Cell i NOS gene m RNA and protein levels of i NOS and RNIs main sterilization product no were significantly up-regulated,but raised by the infection of mouse macrophages is more significant.RNIs pathway blockade electron microscope and laser confocal results show that murine macrophages of Leptospies were swallowed bubble surrounded by a membrane decreased with time prolonged infection,Leptospies morphology is clear and the number gradually increased,intracellular Leptospies and lysosomal membrane were determined gradually reduced,whereas the co localization of THP-1 Cell lysosomes and Leptospies was slightly reduced,a slight increase in the number of Leptospies,suggesting that RNIs pathway in mouse and human macrophages of Leptospies interrogans bactericidal effect.