| Objective:Rheumatoid arthritis(RA)is a systemic autoimmune disease with chronic,aggressive,peripheral arthritis.The etiology and pathogenesis of RA are not completely clear.RA is characterized by synovitis and invasiv pannus formation,as the disease progresses,resulting in the destruction of bone and cartilage,eventually malformation even disability.Angiogenesis is the basis of aggressive pannus formation.Neutrophil extracellular traps(NETs)is released by neutrophils undergoing appropriate stimulation,made up of chromatin fiber decorated with granule proteins and antibacterial peptides,playing roles in the occurrence and development of a variety of diseases.Previous studies have shown that NETs is a sources of citrullinated autoantigens in patients with RA,suggesting that NETs is involved in the development of RA.In chronic obstructive pulmonary disease,NETs can promote pulmonary angiogenesis.Serum amyloid A(SAA)is a kind of acute phase reactive proteins,the previous study found that SAA can induce the formation of NETs.On the basis of previous study,we further explored the role of NETs in angiogenesis in RA and the molecular mechanism of NETs formation induced by SAA.Methods1.The presence of NETs in synovial membrane of RA and OA were detected by immunofluorescence staining.2.Neutrophils were isolated by density gradient centrifugation from healthy subjects,and NETs formation was induced by LPS.After extracting NETs,the concentration of DNA-NETs was measured by nanodrop spectrophotometer.3.The proliferation of HUVECs in vitro induced by NETs was detected by MTT assay.4.The migration of HUVECs in vitro induced by NETs was analyzed by scratch would healing assay.5.The tube formation of HUVECs induced by NETs was detected by the three-dimensional model of tube formation assay in vitro.6.Neutrophils from the peripheral blood of RA patients and healthy subjects were extracted,the purity of neutrophils was determined by HE staining,and DAPI staining was used to observe the nuclear morphology of neutrophils.7.The isolated neutrophils were divided into RA group and HC group,with the following treatment: tris-HCL buffer,LPS,SAA,respectively.The formation of NETs was observed under the microscope,and the percentage of NETs were calculated.8.The peripheral blood neutrophils from healthy subjects were divided into six groups: control,SAA,(SAA+anti-TLR4-Ab),LPS,(LPS+anti-TLR4-Ab)and anti-TLR4-Ab.Appropriated stimulation was performed to each group.The supernatant was collected for the following detection of DNA concentration.Immunofluorescence was used to observe the formation of NETs,and the percentage of NETs were calculated.9.Pico Green kit was used to detecte the concentration of DNA in supernatant.DNA standard solution was prepared with Calf-thymus DNA,and the standard curve was drawn to calculate the concentration of DNA in supernatant.Result1.Immunofluorescence showed that NETs in the synovial tissue of RA patients were more than OA patients'.2.Fluorescence staining showed that LPS could induce NETs formation,and the concentration of DNA-NETs was 27.8mg/L/106 cell.3.MTT assay showed that compared with the control group(0.405±0.445),NETs can promote the proliferation of HUVECs(1.108± 0.012,p<0.05).Then in the pretreatment of NETs with DNase?(0.919± 0.056),the ability of NETs to promote the proliferation of HUVECs was significantly inhibited(p<0.05).4.Scratch would healing assay showed that compared with the control group(16.57 ±1.066%),NETs can promote the migration of HUVECs(78.43 ± 1.784%,p<0.05).In the treatment of NETs with DNase ?(44.57±1.798%),the ability of NETs to promote the migration of HUVECs was significantly inhibited(p<0.05).5.The three-dimensional model of tube formation assay displayed that NETs can promote the formation of HUVECs nodes(2.857±0.3401 vs.1.143±0.4041,p<0.05),pretreatment with DNase ? could inhibit the ability of NETs(1.571±0.3689 vs.2.857±0.3401,p<0.05).6.The purity of neutrophils was more than 95% by HE staining.DAPI staining showed the typical morphology of neutrophil nucleus is band form nucleus and multilobe nucleus.And the purity and the state of the neutrophils were all up to the experimental requirements.7.The fluorescence staining displayed that neutrophils from RA patients were more likely to form NETs than healthy subjects in SAA group(19.10±0.7922% vs.14.67±1.078%,p<0.05)and spontaneous group(7.394±0.5270% vs.4.85±0.3659%,p<0.05),and NETs from RA was more typical.8.The percentage of NETs in SAA group(14.58±1.058%)and LPS group(13.70±0.794%)was significantly higher than the control group(5.734±0.428%,p<0.05).The percentage of NETs in SAA group with the neutralizing antibody pretreatment(6.915±0.418%)was significantly lower than that of SAA group(14.58±1.058%,p<0.05).(LPS+anti-TLR4 Ab)group(6.905±0.4646%)was clearly lower than LPS group(13.71±0.7951%,p<0.05).9.The results showed that the concentration of DNA in supernatant of SAA group(36.89±1.288 ?g/m L)and LPS group(35.23±0.7689 ?g/m L)were significantly higher than that of control group(12.65 ±0.8157 ?g/m L,p<0.05).The concentration of DNA in supernatant of SAA group with TLR4 neutralization antibody pretreatment was obviously decreased(16.34±0.611 ?g/m L,p<0.05).The concentration of DNA in supernatant in(LPS+anti-TLR4-Ab)group(15.77±0.8893 ?g/m L)was significantly lower than that of LPS group(p<0.05).Conclusion:1.Neutrophils in RA patients were more likely to form NETs.More NETs were observed in RA synovial tissue compared with OA synovial tissue.Neutrophils were in the pre-activated state in RA,and their ability to form NETs was stronger than that of healthy control.2.NETs could promote proliferation,migration and lumen formation of HUVECs,which suggests that NETs may promote angiogenesis.After pretreated with DNase?,the ability of NETs promoting angiogenesis was inhibited.The results indicated that DNA of NETs plays an important role in the role of NETs in promoting angiogenesis.3.SAA could induce the formation of NETs by banding to TLR4 in rheumatoid arthritis. |
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