The Effects And Mechanism Of Mycobacterium Tuberculosis PknG On Macrophage Polarization

Objective: To observe the effects of Mycobacterium tuberculosis(Mtb)serine-threonine-protein kinase G(PknG)on macrophage M1 and M2 types and their cytokines,and to explore the effect of PknG on macrophage polarization and its mechanism.Methods: Taking the strain Mycobacterium smegmatis(MS)as the experimental strain,it was divided into two groups: MS(MS::PknG)overexpressing PknG(MS::PknG)group(PknG group)and empty strain(MS::Vector)group(Vector group).The mouse macrophage cell line RAW264.7 was stimulated with MOI=10 for 0 h,12 h and 24 h,and the levels of CD86 and CD206 molecules on the surface of macrophage membrane were detected by flow cytometry;q PCR was used to detect the expression of i NOS,TNF-α,IL-6,IL-12,IL-1β and TGF-β m RNA in cell lysates after stimulation for 0 h,24 h and 48 h;The levels of TNF-α,IL-6,TGF-β and IL-10 in cell culture supernatants for 0 h,24 h and 48 h;Western blot detection of P65 and P38 protein phosphorylation in cell lysates for 0 h,2 h and 4 h after stimulation to observe the effect of PknG intervention on macrophage polarization.Results: The results of flow cytometry showed that after PknG stimulated RAW264.7cells at 12 h and 24 h,compared with the Vector group,the M1 macrophage surface molecule CD86 level in the PknG group increased(P<0.01);q PCR results showed that after stimulating RAW264.7 cells at 24 h,compared with the Vector group,the M1-type cytokine IL-1β m RNA expression was increased in the PknG group(P<0.01),and the M2-type cytokine TGF-β m RNA expression was decreased(P <0.05);After stimulating RAW264.7 cells at 48 h,compared with Vector group,the m RNA expressions of M1-type cytokines i NOS,TNF-α,IL-6,IL-12 and IL-1β in PknG group were increased(P<0.01),and M2-type cytokine TGF-β m RNA expression decreased(P < 0.01);ELISA results showed that after PknG stimulated macrophage RAW264.7 at 24 h,compared with Vector group,the levels of M2-type cytokines IL-10 and TGF-β in the supernatant of PknG group decreased(P<0.05).Western blot results showed that there was no obvious abnormality in the phosphorylation of P38 in the MAPK signaling pathway,and the phosphorylation of P65 was significantly increased.Conclusion: PknG stimulation can promote the transformation of macrophages to M1 type,promote the production of inflammatory cytokines,and provide a theoretical basis for the pathogenic mechanism of Mycobacterium tuberculosis.