Study On Effects And Mechanism Of Iguratimod On Citrullination Of Peripheral Blood Leucocytes From Rheumatoid Arthritis Patients

Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterised by persistent synovitis and progressive damage to articular cartilage and subchondral bone which mediated by autoantibodies,and its etiologyhas not been known clearly.The clinical manifestations range from mild and self-limiting disease to rapidly progressive inflammation with joint destruction and severe physical disability.The serological features are the production of rheumatoid factor(RF)which targeting immunoglobulin(Ig)and anti-citrullinated protein antibody(ACPA)which targeting citrullinated protein(CP).For now,the detailed mechanism of RA is still unknown.ACPAs are considered to play important roles in the pathogenesis and pathological process of RA.ACPAs promote hyperplasia of the synovium and damage of bone and joints by forming immune complexes,and CPs are the main targets of ACPAs.The interaction of abnormally increased CPs and the immune system leads to ACPA generation.Citrulline,a non-essential amino acid,is generated post-translationally by peptidylarginine deiminases/protein-arginine deiminases(PADs),a family of five calcium-dependent enzymes(termed PAD1-4 and 6)that convert arginine residues to citrulline residues.Some evidence indicates that PAD2 and PAD4 play a crucial role in RA.PAD2 and PAD4 are expressed by neutrophils and monocytes,but these two cell types play different roles in RA.Moreover,the effect of RA drugs on PADs is unclear.Iguratimod(IGU)is a new synthetic small-molecule disease-modifying anti-rheumatic drug(DMARD)for the treatment of RA in China and Japan,the safetyand effectiveness of which have been confirmed by post-marketing surveillance.Previous studies found that IGU inhibits the release of bradykinin in rat models of inflammation,and the secretion of interleukin(IL)-6 and IL-8 in RA fibroblast-like synoviocytes(FLS).IGU reduced serum levels of tumor necrosis factor(TNF)-?,IL-1? and IL-6 in rats with collagen-induced arthritis.IGU can also inhibit immunoglobulin(Ig)production by cultured B cells.In addition,IGU is effective in refractory rheumatoid arthritis patients.At present,the effect of IGU on citrullination,a key pathogenic factor of RA,has not been fully studied.Objectives:To investigate whether IGU affects the generation of CPs via PAD and compare the effects of IGU and other therapeutic drugs for RA on TNF-?,IL-1?,IL-6 and IL-8.Furthermore,to study the effects and mechanism of IGU on citrullination of peripheral blood leucocytes in patients with RA in order to provide reference for RA treatment and potential therapeutic targets.Methods:Neutrophils and peripheral blood mononuclear cells(PBMCs)were isolated from ten RA patients who had received no drug treatment except nonsteroidal antiinflammatory drugs(NSAIDs)in the prior year,and the ten patients excluded from infection and other immunity diseases.Then the two type of cells were treated with various concentrations of IGU(5,10,and 20 ?g/ml),methotrexate(MTX;10and 100 nmol/L,nM),or dexamethasone(DXM;10 and 100 ?mol/L,?M)for 8 hours.The cells treated without any drugs except culture medium were considered as control.The cell culture supernatants were harvested and concentrations of TNF-?,IL-1?,IL-6,and IL-8 were detected by enzyme-linked immunosorbent assay(ELISA).Total protein was extracted from the two types of cultured cells,and the expression of CP,PAD2 and PAD4 was detected by western blot.Total RNA was extracted from the two types of cultured cells and used to synthesize cDNA.Quantitative real-timepolymerase chain reaction(qRT-PCR)was performed to evaluate the expression of PAD2 and PAD4 mRNA.Results:IGU inhibits CP and PAD expression in neutrophils.In neutrophils,IGU,MTX,and DXM significantly inhibited the expression of CPs,PAD2,and PAD4 compared with control(P < 0.05),and the inhibition was related to drug concentration.The inhibitory effect of 10 ?g/ml and 20 ?g/ml IGU,100 nM MTX,and 100 ?M DXM was statistically significant compared with the control(P < 0.01).20 ?g/ml IGU decreased CP,PAD2,and PAD4 to 0.77-fold,0.69-fold,and 0.74-fold,respectively.100 nM MTX decreased CP,PAD2,and PAD4 to 0.78-fold,0.71-fold,and 0.71-fold,respectively.100 ?M DXM exhibited the most significant effect(P < 0.05);CP,PAD2,and PAD4 levels were decreased by 0.68-fold,0.46-fold,and 0.53-fold,respectively.And there were no differences in effect between 20 ?g/ml IGU and 100 nM MTX(P > 0.05).In PBMCs,however,the changes of CP,PAD2,and PAD4 expression by IGU,MTX,and DXM ranged from 0.82-fold to 1.18-fold,0.64-fold to 1.14-fold,and0.73-fold to 1.14-fold respectively.But none of these changes had statistical significance compared to control(P > 0.05).IGU inhibits the expression of PAD mRNA in neutrophils.The changes of PAD at mRNA levels similar to those at protein levels.In neutrophils,PAD mRNA expression was significantly decreased by all three drugs compared with that in the control group(P < 0.01),and the inhibition was related to drug concentration.And there were no differences in effect between 20 ?g/ml IGU and 100 nM MTX(P >0.05).In PBMCs,the changes of PAD2 and PAD4 expression by IGU,MTX and DXM ranged from 0.41-fold to 1.82-fold,and 0.40-fold to 1.73-fold respectively.But none of these changes had statistical significance compared to control(P > 0.05).IGU inhibits the secretion of pro-inflammatory factors by neutrophils and PBMCs,and the inhibition was related to drug concentration.The results showed that all three drugs decreased the secretion of TNF-?,IL-1?,IL-6,and IL-8 by neutrophilsand PBMCs compared with control.These effects were statistically significant(P <0.01)at an IGU concentration of 20 ?g/ml,a MTX concentration of 100 nM and at a DXM concentration of 100 ?M.IGU at 10 ?g/ml and MTX at 10 nM also significantly decreased the pro-inflammatory factors in both neutrophils and PBMCs(P < 0.05).The inhibitory effect of 100 ?M DXM on inflammatory factors was the strongest compared to these of 20 ?g/ml IGU and 100 nM MTX(P < 0.05),except in the case of IL-1?.But there were no differences in effect between 20 ?g/ml IGU and100 nM MTX(P > 0.05).Conclusions:1.IGU inhibited citrullinated protein expression in neutrophils from rheumatoid arthritis patients similarly to methotrexate at appropriate concentrations.2.IGU inhibited the expression of PAD at both the protein and mRNA levels in neutrophils from rheumatoid arthritis patients similarly to methotrexate at appropriate concentrations.3.IGU decreased the secretion of TNF-?,IL-1?,IL-6,and IL-8 by neutrophils and PBMCs in rheumatoid arthritis patients,and the inhibitory effect increases with the increase of IGU concentration.