Effects Of Psoriasis Mesenchymal Stem Cell-derived Exosomes On PBMC Secretion Of Cytokines And Proteomic Analysis

BackgroundPsoriasis is a chronic inflammatory skin disease that is prone to recurrence,and currently the helper T lymphocyte(Th17)/regulatory T lymphocyte(Treg)and its associated cytokine imbalance are thought to play a key role in the pathogenesis of psoriasis.Mesenchymal stem cells(MSCs)are matrix cells that exist in a variety of tissues and have the ability to self-renew and exhibit multilineage differentiation.Dermal mesenchymal stem cells(DMSCs)are one of the important components of the skin microenvironment,with differentiation and regeneration functions;It can participate in immunomodulation by secreting cytokines and have an important effect on the skin microenvironment.DMSCs in psoriasis patients perform abnormally in cytokine secretion,DNA methylation and immune regulation,while stimulating abnormal proliferation of epidermal cells and shortening cell cycle.Exosomes(EXO)are epicellular vesicles containing a variety of proteins,m RNA,mi RNA,ceramides and cholesterol,which play an important role in cell communication and transmission of biological information.MSCs-derived exosomes are the primary carriers for transmitting information between them and target cells.Studies have shown that exosomes assume the immunomodulatory role of MSCs,either by carrying biological signals or by directly participating in the activation of certain immunoregulatory pathways.Proteins play an important role in life activities,and proteomics including mass spectrometry detection and bioinformatics analysis can be used to conduct comparative statistical research on the characteristic information of proteins,namely protein expression,post-translational modification,protein interaction,etc.,and improve the overall understanding of genetic biomedicine at the protein level.For emerging exosome research,research on exosome proteomics derived from mesenchymal stem cells for psoriasis skin lesions has not been reported.In this study,the exosomes derived from normal human and psoriasis MSCs were extracted for proteomic analysis,and the peripheral blood mononuclear cells PBMCs of healthy people and psoriasis patients were used to analyze the effect of DMSCs-EXO on the secretion of psoriasis-related factors by PBMCs in PBMCs..Method1.The exosomes were isolated and purified from the supernatant of DMSCs by differential ultracentrifugation.The Mesenchymal stem cell were observed by transmission electron microscopy,Bicinchoninic acid(BCA)and nanoparticle tracking analysis(NTA)were used to determine the size and concentration of exosomes.2.The Ficoll-Hypaque method was used to isolate and culture PBMCs from the peripheral blood of healthy volunteers and psoriasis patients,and psoriasis dermal mesenchymal stem cell-derived exosomes(PDMSCs-EXO)and normal human skin mesenchymal stem cell-derived exosomes(PDMSCs-EXO)were added respectively mesenchymal stem cell-derived exosomes,HDMSCs-EXO),the supernatant was collected before,24 hours and 72 hours after addition,and the psoriasis factors associated with PBMCs were determined by enzymelinked immunosorbent assay(ELISA),IL-17,IL-23,IL-10 and TGF-β1,and the results were statistically analyzed using SPSS26.0 and performed by two-factor repeated ANOVA,with P<0.05 as the difference being statistically significant.3.According to the unlabeled quantitative proteomics experiment,the exosome samples were detected by mass spectrometry,after polyacrylamide gel electrophoresis(SDS PAGE),it was enzymatically hydrolyzed by Filter-Aided Sample Preparation(FASP),and the data were analyzed by Maxquant software.Finally,the results were statistically analyzed to obtain differential proteins and bioinformatics.Results1.The cystic structure of DMSCs-EXO was observed by transmission electron microscopy(TEM),and the diameter of DMSCs-EXO was mainly distributed between30-150 nm by NTA.2.2.ELISA detection showed that PDMSCs-EXO and HDMSCs-EXO acted on normal human peripheral blood mononuclear cells(Healthy PBMCs,HP)and peripheral blood mononuclear cells(Psoriasis PBMCs,PP)of psoriasis patients,respectively,PDMSCs-EXO significantly upregulated IL-17,IL-23 and downregulated IL-10 and TGF-β1(P<0.05),HDMSCs-EXO had significant up-regulation of IL-10 and TGF-β1and down-regulation of IL-17 and IL-23 on PP(P<0.05),and there were statistical differences between different groups of IL-23,IL-17,IL-10 and TGF-β1 in three time points(P<0.001).3.Label Free Mass Spectrometry and bioinformatics analysis with HDMSCs-EXO as a control group,they included seven down-regulated proteins: HTRA3,B2R4D5,MFAP5,FBLN2,CEMIP,GNAQ,MFGE8 and six up-regulated proteins: SCUBE3,Q53G97,Hel-S-83 p,A0A140VJI7,HDLBP,B7Z4M5,its functions and localization are mainly concentrated in the aspects of biological metabolism,inflammation pathway,extracellular vesicle function,cell communication and so on.Conclusion1.PDMSCs-EXO may be involved in transmitting inflammatory signals,affecting PBMCs to secrete pro-inflammatory cytokines and inhibit anti-inflammatory cytokines,catalyzing the occurrence of psoriasis.At the same time,HDMSCs-EXO may be involved in regulating inflammatory responses caused by cytokine imbalance.2.Compared with HDMSCs-EXO,there are 13 differential proteins in PDMSCsEXO,most of which are enriched in various metabolic processes,regulation of inflammatory signaling pathways,physiological functions of extracellular vesicles and other biological processes.Among them,B2R4D5,MFGE8 and MFAP5 may be related to the inflammatory mechanism of psoriasis.