| Objective:To investigate the role and mechanism of SIRT7(sirtuin7)in the infection of host cells by M.tbMethods:Isolate CD 14+monocytes from peripheral blood mononuclear cells(PBMCs)of tuberculosis patients and healthy people,and extract total RNA for reverse transcription,then use real-time fluorescent quantitative PCR to detect changes in SIRT family gene expression;Infect RAW264.7 cells with Mycobacterium tuberculosis H37Rv cultured to logarithmic phase for 24h,respectively,detect the concentration of NO in the supernatant and the difference of CFU by spreading plate method;Infect RAW264.7 cells with BCG-GFP for 24h,and use flow cytometry to detect changes in intracellular bacterial load;Lentiviral packaging and Lipofectamine 3000 liposome transfection were used to obtain SIRT7 knockdown and overexpressing cell lines,which were verified by quantitative reverse transcription PCR(qRT-PCR)and Western blotting;Apoptosis was stained by Annexin-V/PI kit,then detected by flow cytometry;and analyzed by FlowJo software;All datas were analyzed and plotted in GraphPad Prism 6.01,with the mean±standard deviation(X±SD)as statistics,and analysis of variance(ANOVA)or t test.P<0.05 considered statistically significant.Results:(1)By quantitative PCR detection,the expression of SIRT7 in CD14+ monocytes of tuberculosis patients was significantly lower than that of healthy people(3.93±0.28 vs 1.81±0.12,***P<0.001);Similarly,when we infected RAW264.7 cells 24 with H37Rv and tested again,the expression of SIRT7 was significantly reduced compared with no infection(2.86±0.34 vs 1.43±0.07,***P<0.001).(2)Tuberculosis infection can significantly increase the concentration of NO in the supernatant(2.42±0.36 vs 10.92±2.72,**P<0.01);When pre-treated with NAM and re-infected,the concentration of NO in the supernatant decreased(10.92±2.72 vs 2.88±0.40,**P<0.01),further testing the expression of NO-related genes,we found that the expression of iNOS and L-arg also decreased.(3)Compared with the control group,the macrophage treated with NAM at a concentration of 10 mM in advance,the intracellular bacterial load increased significantly(25.03 ± 0.79 vs 39.43±0.67,***P<0.001);when we knocked down at SIRT7 The overexpression cell line was verified again,and the same result was obtained that SIRT7 knockdown can significantly increase the intracellular bacterial load.(4)SIRT7 knockdown can suppress early apoptosis(16.13±1.48 vs 7.58±0.71,***P<0.001);early apoptosis increases after overexpression(3.80±0.30 vs 9.49±1.28,***P<0.001);However,it had no significance on late apoptosis(11.93±0.74 vs 9.49 ±1.28,***P<0.001).(5)We found that CFU count was significantly increased in the group treated with iNOS inhibitor L-NAME and L-NMMA in advance by coating plate method CFU(11.33±1.45 vs 26.67±3.93,*P<0.05),and the group treated with the SIRT7 inhibitor NAM had the same results(11.33±1.45 vs 23.33±4.05,*P<0.05),and this increase in the number of colonies could be compensated by exogenous NO donor SNAP(23.33±4.05 vs 13.33±2.84,*P<0.05).(6)In the same experiment of SIRT7 knockdown and overexpression,we obtained the same result,that is,SIRT7 knockdown CFU was significantly increased compared with no load(22.33±3.66 vs 42.33±2.18,**P<0.01),compared with the unloaded group,SIRT7 overexpression CFU was significantly reduced(28.02±2.20 vs18.33±2.19,*P<0.05).(7)Finally,we detected the expression of cytokines secreted by macrophages,and we found that compared with the empty group,SIRT7 knockdown can significantly inhibit the expression of NF-?B-dependent cytokinesConclusion:Our results indicate that SIRT7 can exert its function against M.tuberculosis infection by regulating NO and apoptosis,and SIRT7 can be used as a potential pharmacological target for the treatment of tuberculosis. |
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