Objective To investigate the effect of neutrophil extracellular traps (NETs) on inflammation of rheumatoid arthritis (RA), especially angiogenesis. Methods The presence of NETs in synovial tissues of RA and osteoarthritis (OA) was observed by immunofluorescence assay. Neutrophils were isolated from peripheral blood of health volunteers. Neutrophils were cultured in vitro, the formation of NETs was observed. NETs were extracted as a stimulating agent. The effects of NETs on the proliferation of human umbilical vein endothelial cells (HUVECs) and synovial fibroblasts (RAFLS) were evaluated by MTT, and which were classified into two groups: HUVECs group and RAFLS group, with the following treatment: control and NETs (0.28 mg/L). Wound repair assay was employed to evaluate the effect on the cell migration stimulated with NETs. The experiment was divided into three groups:control, VEGF (40μg/L VEGF) and NETs (0.28 mg/L NETs). Results (1) Compared with OA, NETs were found more in the synovial tissue of RA. (2) NETs formation was induced by stimulator in vitro. The concentration of extracted NETs-DNA was 27.8 mg/L. (3) MTT assay showed that compared with the control groups, low concentration of NETs (0.28 mg/L) promoted the proliferation of HUVECs (0.499 ± 0.011 vs. 0.393 ± 0.009, P<0.05) and RAFLS (0.266 ± 0.007 vs. 0.192 ± 0.007, P<0.05). (4) It was showed that a significant wound closure induced by low concentration of NETs (0.28 mg/L) was found compared with control. Conclusion Our present study suggests that NETs are found more in the synovial tissue of RA, and low concentration of NETs can promote angiogenesis in RA.

Objective To explore whether serum amyloid A (SAA) can induce the formation of neutrophil extracellular traps(NETs)in neutrophils in vitro. Methods A stable method for inducing NETs formation in vitro was established, in-cluding isolation of peripheral blood neutrophils, cell culture, and NETs formation and observation. The neutrophils were iso-lated from peripheral blood of healthy volunteers. And cells were cultured in vitro and classified into three groups:negative control (NC) group, SAA group and lipopolysaccharide (LPS) group. Following the distinct stimulation in three groups, NETs formation was observed and its percentage was calculated. The concentration of hinstone (h) 3 in supernatant was detected by ELISA. Results The purification and vitality of isolated neutrophils were both more than 95%. The nuclei of neutrophils lost their shape and spread, NETs formation was found. More NETs formation was found in SAA group than that in NC group (P < 0.05). Moreover, the concentration of h3 in supernatant was significantly higher in SAA group than that in NC group (P<0.05). Conclusion SAA can induce the formation of NETs in vitro.

Objective To investigate the neutrophil extracellular traps (NETs) formation and their molecular mechanisms induced by serum amyloid A (SAA) in rheumatoid arthritis (RA).Methods Neutrophils were isolated from peripheral blood of RA and healthy volunteers.① Neutrophils were cultured in vitro,the formation of NETs was observed and their percentage was calculated.② Neutrophils were cultured in vitro,divided into six groups:control,SAA,[SAA+anti-Toll like receptro4 (TLR4)-Ab],LPS,(LPS+anti-TLR4-Ab) and anti-TLR4-Ab.Appropriate stimulation was conducted for each group.NETs formation and their percentages were investigated.The concentration of DNA in supernatant was detected by fluorescent staining.F test and t test were used for statistical analysis.Results ① The purification of isolated neutrophils was higher than 95％.The network which was collocated with the spreading neutrophils nucleus and neutrophil elastase under the microscope,was NETs.In the RA group,the formation of NETs induced by SAA was significantly more than control [(19.1±0.8) vs (7.4±0.5),t=12.30,P＜0.05].② However,after pretreated with anti-TLR4 antibody,NETs formation was significantly less than the SAA group [(5.7±0.4) vs (14.7±1.1),t=7.825,P＜0.05].Moreover,the fluorescence intensity of DNA in supernatant was significantly higher in SAA group than that of anti-TLR4-Ab pretreatment group [(18.7 ±0.7) vs (12.9±0.8),t=5.552,P＜0.05].The concentration of DNA in supernatant of SAA group was higher than that of anti-TLR4-Ab pretreatment group [(36.9±1.3) μg/ml and (16.3±0.6) μ,g/ml,t=14.41,P＜0.05].Conclusion SAA can induce the formation of NETs from neutrophils by binding to TLR4 in RA.