Investigation Of SAA Participating In Inflammation Of RA Through Inducing The Formation Of NETs

Objective: Neutrophil extracellular traps(NETs) is a kind of net work composed by chromatin and the granule proteins adhered on it, and NETs are formed by neutrophils when they are exposed to proper stimulants. After they are formed, NETs play a important role in immune responses and they also take a part in the pathogenesis of many diseases. It has been reported that NETs are abnormally formed in patients of many auto-immune diseases and they may affect pathogenetic condition in many ways. Serum amyloid A is a kind of acute phase protein,like neutrophils, SAA is a important player in immune response. Whether SAA impact the formation of NETs, hasn't be reported, while SAA could impact neutrophils in many ways. It was reported that SAA induce the formation of ROS by neutrophils. The formation of ROS is the key process of NETs formation,and it was said that ROS could induce NETs formation. It was proposed SAA may induce the formation of NETs. The research of NETs in RA is just beginning,only a few of papers have been published, and some resultes in those papers are inconsistent. Then, this study focus on exploring the formation of NETs in RA, and weather the formation of NETs were affected by SAA, and how NETs participate in RA inflammation.Methods: 1. The concentration of MPO and NE in serum were determained by ELISA, and cell free DNA in serum were detected by fluorescence staining.2. Immunefluoresence were used to detect NETs in ST of RA patients and OA patients.3. Perason correlational analyses between the level of MPO, NE in serum of RA patients and corresponding DAS28, ESR, CRP, were performed.4. perason correlational analyses between the levels of MPO, NE and the levels of anti-CCP?RFIg G?RFIg A in serum of RA patients, were performed too.5. MTT were used to analyze the effect of NETs on proliferation of FLS and HUVECs.6. First neutrophils were isolated through density gradient centrifugation, then neutrophils were stained by trypan blue to determine their activity, at last HE stain were performed to determine the purity of neutrophils.7. Neutrophils were distributed into SAA group, LPS group and negative control group, and corresponding induced by SAA, LPS and tris-HCl buffer.8. In order to determain the percent of cells that formed NETs, fluorescence staining were performed. ELISA was used to detect the concentration of h3 in supernatant.Result: 1. The concentration of MPO(4.257 ±0.4840 mg/L)and NE(1.992 ± 0.07389 mg/L) in RA serum are higher than corresponding levels in healthy control serum, MPO(1.054 ±0.1130 mg/L),NE(1.707 ±0.06374 mg/L). DNA level in serum of RA(17.51 ± 1.319 RFU) is lower than that of heathy control(22.68± 0.9035 RFU)(P<0.05).2. Immunefluoresence showed compared to OA patients' ST, more NETs were detected in the ST of RA patients.3. The level of MPO in RA serum is positive correlated to DAS28( R2=0.288, P < 0.05) and ESR( R2=0.365, P < 0.05), while it have no correlation to CRP(R2=0.0256, P > 0.05)(pearson correlation).4. The level of MPO in RA serumhave no correlation to the concentration of anti-CCP, RFIg G and RFIg A in RA serum(pearson correlation).5. NETs could promote the proliferation of FLS and HUVECs.6. HE stain and trypan blue stain showed separated neutrophils with the purity and the vitality higher than 95%.7. The percent of cells that formed NETs in SAA group is significantly high compared to negative control group(SAA:0.168 ± 0.008;NC:0.039 ± 0.006;F=46.08,P<0.05, n=12).8. The concentration of h3 in supernatant of SAA group is significantly high compared to negative control group(SAA: 34.24 ± 0.57;NC:24.03 ± 0.74;F=56.25,P<0.05,n=12).Conclusion: 1. The concentration of MPO and NE in RA serum are higher than that in healthy control serum, remined that the level of NETs in RA serum may also higher than that in healthy people. Immunefluoresence showed compared to OA patients' ST, more NETs were detected in the ST of RA patients. All those remind that, at least, NETs were enhanced formed in ST of RA,2. The positive correlation between the concentration of MPO and DAS28?ESR, reminds that MPO maycorrelated with RA inflammation.. In RAST, more NETs were formed and it promote the proliferation of FLS and HUVECs. Above results remind NETs could take a role in synovial inflammation of RA through promote the proliferation of synovium.3. SAA group like LPS group has a higher percent of cells that formed NETs and a higher concentration of h3 in supernatant, all those results reminder that SAA could induce the formation of NETs.