Screening Of Differential Expression Of Plasma Exosome Proteins In Tuberculosis And Study On The Role Of SAA1 In Regulating Macrophage Infection With Mycobacterium Tuberculosis

Background and Objective: Tuberculosis(TB)is one of the world’s most popular infectious diseases,which seriously threatens people’s life and health.Early detection and treatment of TB can significantly reduce its transmission,morbidity and mortality,and how to better diagnose TB is an important for TB prevention and control.In recent years,exosomes have been widely understood as an emerging signaling medium between cells.The exploration of the bioactive components of exosomes will help us to better screen biomarkers for the detection and evaluation of TB treatment and infection status.In this study,plasma exosomes from TB patients were collected and differential proteins were compared and identified using proteomic techniques,and some key molecules were discovered and their mechanisms in the pathogenesis of TB were explored.Methods: 1.The plasma exocrine bodies of tuberculosis patients and healthy volunteers were extracted,and the proteins were screened by non-standard proteome quantitative technology,bioinformatics analysis,and combined with proteomics database,in order to screen out the differentially expressed proteins related to the pathogenesis of tuberculosis;q-PCR method was used to verify the expression level of differential genes in THP1 cells infected with H37 Ra.2.THP1 cells were infected with H37 Ra of MOI=10,and the protein expression and RNA expression of SAA1 gene were detected by Westernblot and q-PCR methods at 0 h,4 h,6 h,12 h,24 h and 36 h after infection,THP1 cells were infected with H37 Ra,the release of inflammatory cytokines from THP1 cells after H37 Ra infection was detected by q-PCR method,NO production was detected by Griess method,and the migration ability of THP1 cells was detected by Transwell method.SAA1 overexpression plasmid was constructed to transfect cells,to detect the production of cell-related inflammatory factors,NO and cell migration ability,to collect exosomes released from THP1 cells without infection and after H37 Ra infection,to detect exosome marker proteins by Westernblot method,to observe the morphology of exosomes under electron microscope,and to evaluate exosome quality.The exosomes collected were co-incubated with THP1 cells to detect the release of inflammatory factors,the production of NO and the ability of cell migration;after co-incubation with THP1 cells with SAA1 recombinant protein,the inflammatory factors,NO production and cell migration were analyzed,which played a regulatory role.Results: 1.Compared with healthy volunteers,68 differential proteins were detected in plasma exosomes of tuberculosis patients,of which 46 were up-regulated and 24 were down-regulated(P value < 0.05,Foldchange > 1.5).Bioinformatics analysis showed that the differential proteins were mainly related to ECM receptor interaction,tight junction and coronavirus disease-Co VID-19.Compared with the online database,it was found that the six differential proteins were common differential proteins in multiple proteomics,namely SAA1,HBB,HP,APCS,ORM1 and ITIH4.Bioinformatics analysis showed that six proteins were related to cell death,oxidation and detoxification and acute phase response;after THP1 cells were infected with H37 Ra,the expression of SAA1,HBB,HP,APCS and ORM1 was up-regulated,while the expression of ITIH4 was down-regulated.2.The expression of SAA1 in THP1 cells infected with H37 Ra peaked 6-12 h after infection,and then decreased but remained higher than normal.After H37 Ra infection,the m RNA expression levels of inflammatory factors IL-1 β and TNF-α increased,while the expression of i NOS increased,the production of NO increased,and the ability of cell migration enhanced.Overexpression of SAA1 gene can stimulate the expression of IL-1 β and TNF-α,enhance the expression of i NOS,promote the release of NO and improve the ability of cell migration,and co-incubation with SAA1 recombinant protein can also increase the release of inflammatory factor TNF-α and increase the ability of cell migration.After the exosomes released from THP1 cells infected with H37 Ra were co-incubated with recipient cells,the expression of inflammatory cytokines IL-1 β and TNF-α increased,the production of NO increased,and the ability of cell migration enhanced.Conclusions: 1.In the pathogenesis of tuberculosis SAA1,HBB,HP,APCS,ORM1 and ITIH4 may play an important role and participate in the immune response.2.Macrophages infected with Mycobacterium tuberculosis will up-regulate the expression of SAA1,increase the release of inflammatory factors and the production of NO,improve the immune killing ability of cells;at the same time,it will also improve the ability of cell migration and contribute to the recruitment of macrophages.3.After macrophages are infected with Mycobacterium tuberculosis,the release of exosomes can increase the release of inflammatory factors,the production of NO and the ability of cell migration,in which SAA1 protein may play a regulatory role.