| ObjectiveIdentification of the proteomic and miRNA expression profile of exosomes derived from syphilis patient plasma,and exploration of the role of extracellular vesicle miR-197-3p in Tp-induced macrophage inflammation and its underlying mechanisms.MethodsPlasma samples were collected from 3 patients with secondary syphilis and 3 healthy controls.Plasma exosomes were purified using differential centrifugation-ultracentrifugation and characterized by western blot,transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).Protein profiling was performed using label-free Mass spectrometry.Differentially expressed proteins were identified using a fold change cutoff of 1.5 and a p-value cutoff of 0.05.Functional annotation and pathway enrichment analysis of differentially expressed proteins were conducted using the Database for Annotation,Visualization and Integrated Discovery(DAVID).To validate the protein profiling results,three target proteins(MMRN1,COL6A1,and CRISP3)were selected and measured by enzyme-linked immunosorbent assay(ELISA)in a larger sample size of 18 patients with SS and 16 volunteers as HC.The diagnostic sensitivity and specificity of exosomal proteins were evaluated by receiver operating characteristic(ROC)curve analysis.Moreover,blood samples from serofast status,secondary syphilis,serologically cured patients and healthy people were collected,then exosomal miRNAs were extracted.Microarray analysis of miRNAs was used to identify the miRNA expression profiles of serofast status,to find out differentially expressed miRNAs and their target genes.After that,GO and KEGG were used to analyze the functions and pathways they involved in.Finally,we expanded the sample size to validate the selected miRNAs by RT-qPCR and assessed their diagnostic sensitivity and specificity.Through cell experiments in vitro,miR-197-3p mimics transfected cell models and lentiviral knockdown Syk macrophage models were constructed,and their functions in Tp-induced macrophages pro-inflammatory response were detected by using immunofluorescence,Western Blot,RT-qPCR,ELISA,flow cytometry,and other methods,verifying the transcript levels and cell supernatant secretory expression levels of cytokines such as IL-1β,IL-6,TNF-α,IL-12,and TGF-β,as well as expression levels of inflammasome NLRP3 and macrophage M1-type polarization markers CD86,iNOS and M2-type polarization marker CD 163,while verifying activation of NF-κB pathway,altered p65 phosphorylation,and NF-κB p65 nuclear translocation.The relationship between Syk and miR-197-3p was verified by bioinformatics analysis and dual luciferase reporter assay,and then we verify whether Syk is involved in the regulation of Tp-induced inflammatory response in macrophages.To further investigate the regulatory mechanism of Syk in macrophage inflammatory response,we analyzed the downstream mechanistic pathway of Syk using mRNA-seq and performed preliminary validation.ResultsPart ⅠProtein expression characteristics of plasma exosomes in secondary syphilis patientsA total of 463 proteins were identified by label-free mass spectrometry analysis of the isolated exosome fractions,of which 56 were upregulated and 15 were downregulated in SS.These proteins were involved in various biological processes,such as receptor-mediated endocytosis,immune response,complement activation,coagulation,and antibiotic biosynthesis.The functions of the identified proteins were related to various pathways,including receptor-mediated endocytosis,immune response,and complement and coagulation cascades.The study also confirmed high expression of MMRN1 and low expression of COL6A1 and CRISP3 in plasma exosomes of SS using ELISA,suggesting their potential role as markers for diagnostic screening and prognostic indication of syphilis infection and pathogenesis.Part ⅡPlasma exosomal miRNA expression characteristics in serofast status patientsUsing microarray sequencing technology,the differentially expressed microRNAs(DEmiRNAs)of plasma exosomes from serofast status patients were identified,and it was found that the miRNA expression profiles of serofast status and healthy groups were similar,with only one miRNA expression difference.The differences with the secondary syphilis were larger,with 44 differentially expressed miRNAs.Compared with a serologically cured patient,only 11 miRNAs were expressed with significant differences.Functional annotation of the target genes of the 11 miRNAs revealed that they were associated with a series of biological processes,including transcriptional regulation,regulation of mitochondrial and Golgi structure and function,immune system function,endocrine system function,apoptosis,VEGF signaling pathway,and p53 signaling pathway.The differential expression of miR-197-3p,miR-1908-3p,miR-1273g-3p and miR-4485-5p in plasma exosomes from serofast status patients was verified by RT-qPCR after expanding the sample sizes,and the combination of these four miRNAs was found to further improve the sensitivity and specificity of differentiating serofast status from serologically cured compared with individual miRNAs.Part ⅢMicroRNA-197-3p inhibits Tp-induced macrophage inflammatory response by targeting SykThrough in vitro cellular experiments,it was verified that in vitro cultured macrophages can internalize and ingest exogenous exosomes;and co-culturing exosomes from serofast status patients with macrophages can increase the expression of miR-197-3p in macrophages.Then,we used the miR-197-3p mimics to transfect macrophages to focus on the function of miR-197-3p.After transfection of the miR-197-3p mimics,the transcription level and secretion expression of pro-inflammatory cytokines induced by Tp in macrophages were inhibited to different degrees.The expression of NLRP3,iNOS,and CD86 decreased,while the activation of the NF-κB pathway was inhibited,and the nuclear translocation efficiency of NF-κB p65 decreased.Through multiple database analysis and dual luciferase reporter assay,Syk was verified as the target gene of miR-197-3p,and the transfected miR-197-3p mimics macrophages had low expression of Syk and the phosphorylation level decreased.Subsequently,we constructed shSyk lentivirus and successfully knocked down the expression of the Syk in macrophages.After knocking down Syk,the transcription level and secretion expression of pro-inflammatory cytokines induced by Tp in macrophages were inhibited,and the expression of NLRP3,iNOS,and CD86 decreased,while the activation of the NF-κB pathway was inhibited,and the nuclear translocation of NF-κB p65 decreased.To further explore how Syk participates in macrophage inflammatory response,we used mRNA sequencing to analyze the downstream mechanism pathway of Syk,and preliminarily verified that the Syk participates in the activation of PI3K pathway.Conclusion1.Plasma exosome MMRN1,COL6A1,CRISP3 may play an important role in the pathogenesis of syphilis,and have the potential as biomarkers for diagnosing syphilis and indicating prognosis.2.The differentially expressed miRNAs in plasma exosomes isolated from serofast status patients are involved in various biological processes,including regulation of transcription,immune system,apoptosis,VEGF signaling pathway,and p53 signaling pathway.Among the identified miRNAs,miR-197-3p,miR-1908-3p,miR-1273g-3p,and miR-4485-5p showed some diagnostic efficacy in differentiating serofast status from serologically cured patients or healthy individuals.The study suggests that plasma exosomal miRNAs have the potential to be used as novel biomarkers for serofast status.3.Exosomal miR-197-3p inhibits the Treponema pallidum-induced macrophage inflammatory response by targeting Syk.It also inhibited M1 polarization of macrophages,reduced the expression of NLRP3 inflammasome,inhibited the activation of the NF-κB pathway,and decreased the secretion of pro-inflammatory cytokines.The study suggests that the PI3K pathway may be involved in the regulation of downstream inflammatory pathways by Syk,and this explains the possible mechanism of Tp immune escape and the occurrence of serofast status. |
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