A Preliminary Study On The Pathogenesis Of Neutrophil Extracellular Traps In Rheumatoid Arthritis

Objective: To Explore the potential effects of Neutrophil extracellular traps(NETs)on rheumatoid arthritis synovial fibroblasts(RA-FLSs)and synovial macrophages in patients with rheumatoid arthritis..Methods: The synovial tissues of RA patients were isolated and cultured from RA patients and identification by immunofluorescence staining.Macrophages were obtained and cultured by mouse peritoneal stimulation test.Peripheral blood neutrophils were extracted from healthy volunteers and used to stimulate NETs’ formation,following with NETs’ extraction.MTS proliferation assay was used to evaluate the effect of NETs on the proliferation of RA-FLSs.ELISA was used to determine the the levels of interleukin-6(IL-6)and interleukin-25(IL-25)in cell supernatant after NETs-stimulated RA-FLSs for 60 h,QRT-PCR was used to determine the expression of connective tissue growth factor(CTGF)m RNA in cells treated with NETs-stimulated RA-FLSs for 60 h.WB was used to determine the expression changes of mammalian sterile 20-like kinase 1(MST1).the supernatant collected after NETs stimulated RA-FLS for 60 h and added to macrophages for 7 days,then the morphological changes were observed by Wright-Giemsa ’ s stain.The results were processed using paired sample t-test and one-way ANOVA.Results: 1)The isolated and purified neutrophils could form NETs by stimulated in vitro.The concentration of extracted NETs-DNA was 58.5ng/ul(1×106 cells).2)Compared with the control group(0ul NETs),NETs can promote the proliferation of RA-FLSs.With the increase of NETs’ concentration,the proliferation of RA-FLSs was also enhanced(F=99.519,P<0.001).3)Compared with the control group(0ul NETs),10 ul NETs can significantly promote the proliferation of RA-FLSs(t =-12.226,P<0.01).4)Pretreatment on NETs with DNase I inhibited its effect on promoting the proliferation of RA-FLSs(t =-2.376,P=0.049).5)NETs stimulated the up-regulation of CTGF m RNA expression in RA-FLSs(30.696 ± 0.468,t =12.13,P<0.01).6)Compared with the control group,NETs could stimulate the down-regulation of MST1 kinase expression in RA-FLSs(P<0.01).7)The concentration of IL-6 and IL-25 in the supernatant of NETs stimulated RA-FLSs was higher than that in the control group(P<0.05).8)The NETs stimulated RA-FLS after 60 h.The supernatant was added to macrophages for 7 days,and a part of macrophages were differentiated into multinucleated giant cells.However,multi-nuclear giant cells were not found in the cultured synovial macrophages with RA-FLSs supernatant for 7 days.Conclusion: NETs can promote the proliferation of RA-FLSs and stimulate the down-regulation of MST1 kinase in RA-FLSs,the up-regulation of CTGF m RNA in RA-FLSs in vitro.The concentration of IL-6 and IL-25 in cell supernatant is higher than that in the control group,suggesting that NETs may be regulated.MST1 kinase mediates proliferation of RA-FLSs,produces CTGF,and releases inflammatory cytokines.The supernatant collected after NETs stimulated RA-FLS 60 h induced macrophage differentiation into multinucleated giant cells,it is the osteoclasts possibly osteoclasts.