| Rheumatoid arthritis(RA)is a systemic autoimmune disease charactered with chronic and progressive synovitis,and high morbidity and mortality rates.The prevalence rate in China is 0.35-0.45%,with a cumulative population of more than 6million people.Its pathogenesis has not been fully elucidated.It is currently believed that the self-protein of genetically susceptible triggered by environmental factors,is modified by peptidyl arginine deiminase Type 4(PAD4)as so called citrullination,abnormally released or overexpressed.It breaks autoimmune tolerance and stimulates the production of anticitrullinated protein antibodies(ACPAs),which in turn triggers immune inflammation of the synovium,resulting in damage to joints and their appendages.Recent studies have found that polymorphonuclear neutrophils(PMNs)and neutrophil extracellular traps(NETs)in RA patients may be one of the most important mechanisms for the formation and abnormal release of citrullinated self-antigens.The programmed death process of neutrophils by releasing NETs is called NETosis,and activated PAD4 is also involved in the process of NETosis.Objective:This study aimed to silence PAD4 in peripheral blood PMNs of RA patients by small interfering RNA(siRNA),and further by inhibiting PAD4-mediated NETosis,to observe its interaction with peripheral blood mononuclear cells(PBMCs)in Transwell co-culture system.To detect the changes of RA synovial-like fibroblasts(FLS)activity and function stimulated by co-incubated supernatant.So as to elucidate the role of NETosis in the pathogenesis of RA and lays the foundation for the development of RA biotherapeutic technology targeting NETosis.Methods Transwell mixed cell culture system was used to co-incubate PAD4-silenced PMNs with PBMCs of RA patients,and then the supernatant was used to stimulate RA-FLS.Immunomagnetic beads were used to detect cytokines in supernatant.The concentration of each subtype anti-cyclic citrullinated peptide(CCP)antibody in co-culture supernatant was analyzed by enzyme-linked immunosorbent assay(ELISA).The Th17/Treg cell ratio was detected by flow cytometry(FCM).The cell proliferation ability of FLS was determined by thiazolyl blue(3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium broide,MTT)colorimetric method.The cell cycle was detected by a cycle detection kit.The FLS cytoskeletal expression was observed after phalloidin staining.The activity of RA-FLS was detected by cell counting kit-8(CCK-8).Transwell chamber method was used to determine the migration ability of FLS.The effects on RA-FLS activity and fuction were compared among the groups of siRNA silencing RA-PMNs PAD4,NETosis selective inhibitor gallic acid(GA)tand tumor necrosis factor inhibitor(TNFi)treatment from upper multiple perspectives.RESULTs:1.Compared with the blank treatment control group,the supernatant IL-1β,IL-6,IL-17 A,TNF-ɑ and matrix metalloproteinase-9(MMP-9)were significantly inhibited in the Nets-targeted treatment groups including the RA-PMNS PAD4 silenced group or GA treatment group(P<0.05).Compared with the RA-PMNs PAD4 silenced group,the above cytokines,as well as in the other inflammatory cytokines such as IL-18,IL-23 and IFNγ,which did not significantly declined in the the NET-targeted treatment,were significantly inhibitored in the TNFi treatment group.2.Compared with the blank control RA group,the TNFi treatment group did not significantly reduce the concentration of anti-CCP antibodies of each subtype(Ig A anti-CCP: P=0.23;Ig G anti-CCP: P=0.21;Ig M anti-CCP: P = 0.65),while the treatment targeting NET including GA treatment group or PAD4 silenced group,can significantly reduce the supernatant concentration of each subtype of anti-CCP antibody(each proup compared with the blank,P<0.01).3.Compared with the normal healthy control group,the proportion of Th17 cells in RA patients was significantly increased(4.17±0.45 和 1.48±0.57),(both P<0.01),and there was an imbalance of Th17/Treg.Compared with the RA blank control group(4.17±0.45),The ratio of Th17 cells was significantly reduced in the PAD4 silenced group(1.63±0.45,P<0.05)or the inhibitor GA treatment group(1.69±0.58,P<0.05),while the ratio of Treg cells did not change significantly.But it is the significantly increased in the TNFi treatment group(1.70±0.44 vs 4.84±1.12,P<0.05).4.The inhibition rate of FLS proliferation in the PAD4 silenced group,GA treatment group and TNFi treatment group(respectively35.36±4.39,31.54±6.59,59.98±8.82)was significantly different from RA blank control group(-29.00±7.78)(both P<0.01),and the proliferation inhibition rate of TNFi group was significantly higher than that of GA treatment group and PAD4 silenced group(P<0.01).Cell cycle ratio assay showed that in comparison with RA blank control group(10.78±8.39),the PAD4 silenced group(37.41±12.10),the TNFi treatment group(47.94±5.92)and the GA treatment group(38.34±0.80)showed significant G1 phase arrest(P<0.01).5.The percentage of cell viability was determined by CCK-8 method.Compared with RA blank control group(1.04±0.30),the GA treatment group(0.54±0.15)and the TNFi group(0.54±0.08)could significantly inhibit the activity of FLS(both P<0.05).The PAD4 gene silenced group had no significant effect on the activity of FLSs(P=0.96).6.Transwell room test was used to detect the migration and invasion ability of FLS cells.The migrated FLS cells count showed that when compared with the blank control group(39.20±3.83),the PAD4 silenced group(14.20±4.27),the GA treatment group(16.80±5.54)and the TNFi treatment group(39.20±3.83)can significantly reduce the migration ability of FLS.Conclusion:By inhibition of NET formation with siRNA silencing PAD4 gene of RA PMNs,the Th17/Treg imbalance in peripheral blood of RA patients could be corrected,the production and release of anti-CCP antibodies and inflammatory cytokines IL-1β,IL-6,IL-17 A,TNFα,MMP 9 could be reduced,which in turn ameliorated the proliferation,activation and invasion of synovial fibroblast-like cells. |
PDF Full Text Request