| objective:To explore the relationship between serum amyloid A(SAA),matrix metalloproteinase-3(MMP-3)and disease activity in patients with rheumatoid arthritis(RA),and to explore the difference of exosomes concentration in circulating blood of patients with different disease activity,and the correlation between exosomal amyloid A and disease activity,so as to find it as a basis for evaluating disease progression.To study the immune activity of circulating exosomes in patients with different disease activity and its role in autoimmune process,and to speculate the relationship between circulating exosomal amyloid A and its pro-inflammatory potential under different disease activity,to reveal the possible mechanism of inflammatory diffusion exosomal pathway in RA,and to provide a new diagnostic strategy for evaluating the course of disease and predicting the efficacy of medication in patients with RA.Methods:The sera of patients with rheumatoid arthritis were collected and the disease activity of all patients included in RA was evaluated by DAS28-ESR.The patients were divided into two groups: DAS28-ESR ? 2.6 as clinical remission group(CR group)and DAS28-ESR > 2.6 as active group(n-CR group).The contents of serum amyloid A and MMP-3 in the two groups were detected by ELISA,the serum exosomes was extracted by differential ultracentrifugation,the surface marker protein CD63 and TSG101 weredetected by transmission electron microscope,the particle size and concentration of exosomes were analyzed,the concentration of protein in heavy suspension sample was detected and the exosomes protein was quantified,The content of amyloid A in exosomes was detected by ELISA.The peripheral blood mononuclear cells(PBMCs)of healthy people were isolated by Ficoll-density gradient centrifugation,the proportion of living cells was detected by trypan blue staining,and the cells culture medium without exosomes was prepared.The circulating exosomes of the patients in the above two groups were incubated with PBMCs for 24 hours.The supernatant was collected and the supernatant cultured for 24 hours without exosomes stimulated was used as the blank control group,Interleukin 6(IL-6)and Interleukin 8(IL8)were detected by ELISA.Result:1.The level of serum amyloid A and MMP-3 in n-CR group were significantly higher than that in CR group(P < 0.0001)2.Correlation analysis of clinical indexes showed that serum amyloid A was positively correlated with erythrocyte sedimentation rate(ESR)(? =0.771,P=0.000),serum amyloid A was positively correlated with C-reactive protein(CRP)(? = 0.601,P=0.000),and serum amyloid A was weakly correlated with rheumatoid factor(RF)(? = 0.343,P=0.003).There was a significant correlation between serum amyloid A and MMP-3(? = 0.776,P =0.000).There was no significant correlation between MMP-3 and RF(? =0.204,P=0.086),but there was a significant positive correlation between MMP-3 and ESR(? = 0.726,P=0.000)and between MMP-3 and CRP(? =0.601,P=0.000).3.The exosomes was extracted from the serum of patients with rheumatoid arthritis,The exosomal surface markers CD63 and TSG101 were verified,and it was verified that the diameter of the exosomes was about148 nm,and the particle concentration was about 1.0E+11 Particles/m L.Theconcentration of serum exosomal protein in active patients[(4.32 ±2.28)?g/?L]was significantly higher than that in clinical remission patients[(1.97±0.87)?g/?L,P < 0.0001],and the concentration of exosomal amyloid A in n-CR group [(47.73 ±29.82)ng/m L]was significantly higher than that in CR group[(16.61±18.58)ng/m L,P<0.0001].There was a significant positive correlation between serum amyloid A and exosomal amyloid A in patients with RA(? = 0.601,P=0.000),and there was a significant positive correlation between exosomal amyloid A and serum CRP(? = 0.571,P=0.001).4.The IL-6 production of PBMCs induced by exosomes in n-CR group[(193.96±100.94)pg/m L] was higher than that in CR group [(86.07±111.06)pg/m L,P=0.01].There was no significant difference in IL-6production between exosomes-induced PBMCs in CR group and control group without exosomes stimulated [(20.85 ±21.73)pg/m L,P>0.05].There was no significant difference in the production of IL-8 in PBMCs induced by exosomes in n-CR group [(48.75±37.63)pg/m L] and in CR group [(29.65±19.83)pg/m L,P>0.05],but the IL-8 induced by exosomes in n-CR group was significantly higher than that in blank control group [(11.45±5.87)pg/m L,P=0.031],the production of IL-8 in PBMCs induced by exosomes in CR group was significantly higher than that in blank control group(P =0.0083).Conclusion1.The level of circulating exosomal amyloid A is related to disease activity in patients with RA,and exosomal amyloid A is significantly correlated with serum amyloid A and CRP,which may be used as a new biomarker to evaluate the disease progression of RA patients.2.The circulating exosomal of patients with RA has immune activity,its pro-inflammatory effect is related to disease activity,exosomal amyloid A may play a role in immune regulation of rheumatoid arthritis. |
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