| Background:Rheumatoid arthritis(RA)is a systemic autoimmune inflammatory disease characterized by the production of autoantibodies and inflammatory factors after the cells involved in the innate and adaptive immune response process are infiltrated and activated in the affected joint.T cells,B cells,The function of neutrophils and macrophages is mainly activated,interacting with fibroblasts,chondrocytes and bone cells inherent in joint tissues.These cells interact with each other in terms of immune recognition,immune defense and immune tolerance,causing a series of complex inflammatory and immune response processes in cartilage,tendon,ligament,bone and other tissues,leading to joint swelling,pain and limited movement,and ultimately may lead to bone damage and joint space narrowing or distortion and other adverse events.As mentioned above,the antibody formed by the body to recognize citrullinated protein(CP)is the anti-citrullinated antibody ACPA.In RA,ACPA induces synovial hyperplasia,bone and joint destruction and increased inflammation by forming immune complexes with citcitine-containing autoantigens.Therefore,ACPAs is considered to be a key pathogenic factor of RA,which also has an important impact on the diagnosis and progression of the disease.CP is the main target antigen recognized by ACPA.Citrulline is a nonstandard amino acid in vivo.It is formed during the post-translational modification of proteins from arginine residues to citrulline residues.This process is known as citrullination of proteins,and the enzyme that plays a key catalytic role in this process is known as peptidyl arginine deiminases (PADs).The PAD family consists of five highly similar isoenzymes(PAD1,2,3,4,and 6),of which four isoforms(PAD1-4)have catalytic activity,while PAD6 loses enzyme activity due to mutations in the active site.PAD4,mainly expressed in peripheral blood neutrophils,is the most important subtype of PAD in the citrullination process of neutrophils,and its gene locus is also the susceptibility gene locus of rheumatoid arthritis.In conclusion,PAD4 plays an important role in RA pathology,but the current studies on the influence of PAD4 expression and activity are still incomplete.With the further study of the pathological mechanism of RA,the role of neutrophils in RA has attracted more and more attention.A large number of activated neutrophils were found in synovial tissue and synovial fluid of joints in patients with RA.If the process of neutrophil anti-inflammatory response is not effectively controlled,the inflammatory response will be amplified and cause tissue damage.One of the prominent manifestations of neutrophils in inflammatory joints is oxidative stress caused by respiratory eruption.nicotinamide adenine dinucleotide phosphatase oxidase,NADPH oxidase will be activated massively in oxidative stress,and its activation can promote the generation of reactive oxygen species(ROS).Studies have shown that ROS affects the formation of NLRP3 inflammatory bodies(NOD-,LRRand pyrin domain-containing 3,NLRP3)in RA macrophages and synovial fibroblasts,but there are few studies on RA neutrophils.As an important signaling molecule in cells,calcium ion plays an important role in cell migration and activation.Studies have shown that after neutrophil activation,a large number of extracellular calcium ions flow into the cell to activate calcium ion related reaction processes.Therefore,we wanted to explore whether the calcium ion concentration required for PAD4 activation,as a key enzyme in ACPAs formation,is related to the oxidative stress of neutrophils.Whether oxidative stress can promote intracellular calcium ion flow through activation of some ion channels,thus activating PAD4 and accelerating citrullination.However,there are few studies on the signaling pathway of calcium ions in neutrophils of RA patients.In recent years,with in-depth research on transient receptor potential channels(TRPs)of neutrophil granulocytes,it has been confirmed by many studies that their non-selective cation permeability function plays an important role in mediating the flow of calcium and sodium ions in and out of cells.In addition,its subtype TRPM2 channel is widely expressed in neutrophils and has ROS sensing performance,but its significance in RA neutrophilinduced inflammation/ citrullination remains unclear.Therefore,we set out to investigate whether TRPM2 plays a role in mediating calcium influx in neutrophil granulocytes in the inflammatory environment,thereby influencing the subsequent pathological effects associated with ROS and calcium,namely,the inflammatory/citrullination effect of neutrophil granulocytes.Iguratimod(IGU)is a novel small-molecule antirheumatic drug,which is widely used in the treatment of RA in China and Japan.In the course of clinical administration,it was found that ACPAs of patients were significantly reduced after the application of Iguratimod,which also had regulatory significance on inflammation-related indicators.Therefore,we wanted to explore whether Iguratimod had regulatory effects on the formation process of CP,the antigen identified with ACPA,and the inflammatory response.Therefore,we applied IGU to detect whether it has regulatory effects on signaling pathways and molecules during oxidative stress of neutrophils.Aims:The purpose of this study was to investigate the changes of NADPH oxidase/ROS during the oxidative stress activation of neutrophils and its effect on the inflammation and citrullination of RA neutrophils.To investigate the effect of TRPM2 on the change of intracellular calcium ion concentration of neutrophils,and to clarify the effect of "ROS/TRPM2/Ca2+" pathway on the inflammation and citrullination of neutrophils.The role of Iguratimod as a new slow acting antirheumatic drug in anti-inflammatory/anti-citrullination was also discussed.Research methods:1.Preliminary exploration on the inflammatory/citrulline-inducing effect of peripheral blood neutrophils in patients with RA and the therapeutic effect of Iguratimod.In this study,10 RA patients who met the 2010 ACR/EULAR RA classification standard and scoring system and did not take any immunosuppressive drugs and 10 healthy subjects were included as the control(healthy control,HC).Peripheral blood neutrophils were collected and isolated from the subjects.The cells were divided into control group,RA group and RA+IGU group and cultured in incubator at 37℃ and5% CO2 for 8h,respectively.Western blot(WB)was used to detect the expression of NADPH oxidase component NCF1,which was associated with oxidative stress.The migration rate of neutrophils in each group was detected by Transwell migration assay.The inflammatory markers of IL-1β and IL-18 in the supernatant of cell culture were determined by ELISA.The expression of NLRP3 inflammasomes associated with inflammation was detected by WB.The expression levels of citrulline-related PAD4 protein and CP protein were detected by WB,and the enzyme activities of PAD4 in each group were measured by ELISA.2.HL-60 cell lines were selected and induced to differentiate into neutrophil-like granulocytes,and LPS was used to stimulate the construction of neutrophil-induced inflammatory/ citrullination-induced model in an inflammatory environment HL-60 cell lines were differentiated into differentiated d HL-60(d HL-60)cells by complete medium containing 1.25%DMSO,i.e.,neutrophil-like cells.Different concentrations of LPS were used to stimulate d HL-60 and incubated in incubator for8 h to construct an inflammatory cell environment,and the results were compared with those of control group without LPS stimulation.The experimental groups were:control group,10ng/ml LPS+d HL-60,100ng/ml LPS+d HL-60,1000ng/ml LPS+d HL-60.According to the method in "1",various indicators were tested to evaluate whether the inflammation/citrullination model was established,and the appropriate LPS stimulation concentration was selected according to the cell activity measured by MTT method.3.Effects of oxidative stress of NADPH oxidase /ROS axis on inflammation/ citrullination of neutrophils via TRPM2 d HL-60 cells stimulated by 100ng/ml LPS were selected and divided into 4groups according to the experimental purpose: d HL-60 group,LPS +d HL-60 group,LPS +d HL-60+VAS-2870 group(VAS-2870 as NADPH oxidase inhibitor),LPS+d HL-60 +ACA group(ACA as TRPM2 blocker),were cultured in incubator for8 h.And test all indicators according to the method in "1".In addition,WB was used to detect TRPM2 expression.The levels of reactive oxygen species(ROS)and free calcium ions in each group were detected by fluorescence microscopy and flow cytometry.4.Iguratimod affects the inflammatory/citrulline-induced activity of neutrophils by regulating the NADPH oxidase /ROS axis d HL-60 cells stimulated by 100ng/ml LPS were selected and divided into 6groups according to the experimental purpose.d HL-60 group,LPS group,LPS+NADPH oxidase inhibitor(VAS-2870)group,LPS+ VAS-2870+ IGU group,LPS+TRPM2 inhibitor(ACA)group,LPS+ +IGU group,and all indexes were detected according to the method in "3".In addition,q RT-PCR was used to detect m RNA expression levels associated with inflammation and citrullination.Results:1.Oxidative stress of peripheral blood neutrophils in patients with RA and its anti-inflammatory/citrullination-inducing effects,Iguratimod has anti-inflammatory /anti-citrullination-inducing effects.The expression of phosphorylated protein of NADPH oxidase component NCF1 in peripheral blood neutrophils of RA group and normal control group and the amount of ROS indicated the existence of oxidative stress.Through the analysis of peripheral blood neutrophils migration rate,supernatant IL-1β,IL-18,and inflammatory related protein NLRP3 inflammatory body expression in RA group and normal control group,it was indicated that neutrophils played an inflammatory role in RA inflammatory response.The citrullination-inducing effect of RA neutrophils was demonstrated by analyzing the expression levels of citrullination-related proteins PAD4 and CP and the changes of PAD4 enzyme activity.The above indexes in RA group were lower than those in untreated RA group,and the difference was statistically significant,indicating that iguratimod had anti-inflammatory/anti-citrullination effects.2.The inflammatory/citrulline-inducing model of RA neutrophils was simulated by LPS stimulation of neutrophils The expression of the phosphorylated protein of NADPH oxidase component NCF1 and the amount of ROS in the LPS-stimulated d HL-60 cell lines showed that the oxidative stress response was enhanced with the increase of LPS concentration.By analyzing the cell migration rate,the expression levels of IL-1β,IL-18 in the supernatant and NLRP3 inflammation-related protein,the oxidative inflammatory response was enhanced with the increase of LPS concentration,and the simulated RA neutrophil-induced inflammation model was established.The expression of citrullination related proteins PAD4 and CP and the change of PAD4 enzyme activity in d HL-60 cell line neutrophils stimulated by different concentrations of LPS showed that citrullination was enhanced with the increase of concentration of low concentration LPS,but there was no difference between 100ng/ml and 1000ng/ml citrullination.The results showed that the simulated RA neutrophil-induced citrullination model was established.Cell viability was determined by MTT assay,which showed that cell viability was reduced by 1000ng/ml LPS compared with other concentrations,so 100ng/ml LPS was selected as the appropriate concentration.3.The NADPH oxidase /ROS axis of neutrophils affects NLRP3 inflammation through ROS/TRPM2/Ca2+Formation of corpuscles and inflammatory mediators and citrullination By comparing the NADPH oxidase inhibitor group with LPS group,the levels of NADPH oxidase components NCF1 and ROS were analyzed,indicating the importance of NADPH oxidase on oxidative stress of neutrophils.By analyzing the cell migration rate,the expressions of IL-1β,IL-18,NCF1 and NLRP3 inflammatory bodies in the supernatant,and the changes of intracellular ROS and Ca2+concentrations in the two groups compared with d HL 60 cells,The results indicated that inhibition of NADPH oxidase expression could decrease the concentration of ROS and Ca2+,and the increase of NLRP3 inflammation-related mediators.The expression level of TRPM2 channel-activated ligand protein ADPR was also lower than that of LPS group.The expression levels of citrullination-related proteins PAD4 and CP and the activity of PAD4 enzyme in neutrophil were also decreased with the inhibition of NADPH oxidase.The expression levels of NCF1,ROS,ADPR and PAD4 in the TRPM2 blocker group and LPS group were compared.There were no differences in the above indexes between the two groups,but the intracellular Ca2+concentration,NLRP3 and inflammatory mediators,and the activities of CP and PAD4 enzymes in the TRPM2 blocker group were lower than those in the LPS group.These results suggest that the NADPH oxidase /ROS axis may influence inflammation and citrullination through TRPM2-mediated Ca2+concentration increase.In addition,the expression levels of NLRP3 and inflammatory mediators inhibited by NADPH oxidase were lower than those in the TRPM2 blocker group,indicating that both ROS and Ca2+played a regulatory role in the formation of NLRP3 in neutrophils.4.Iguratimod regulates neutrophil-induced inflammation/ citrullination by influencing the NADPH oxidase /ROS axis of neutrophils to regulate the ROS/TRPM2/ Ca2+pathway By comparing and analyzing the detection indexes of control group,LPS,LPS+NADPH oxidase inhibitor,LPS+NADPH oxidase inhibitor + IGU,LPS+TRPM2 channel blocker and LPS+ IGU group,By analyzing the changes of NCF1,ROS,ADPR,Ca2+concentration,PAD4 protein and m RNA,CP and PAD4 enzyme activity in LPS+ IGU and LPS groups,the results indicated that IGU inhibited the oxidative stress and inflammatory/citrulline-inducing effect of neutrophils.By analyzing the changes of the NADPH oxidase /ROS axis in LPS+NADPH oxidase inhibitor and LPS+NADPH oxidase inhibitor + IGU group,the addition of Iguratimod further inhibited the amount of NADPH oxidase component NCF1 and ROS,and the expression of Tr PM2-activated ligand protein ADPR also decreased.The results indicated that Iguratimod inhibited the NADPH oxidase /ROS axis of neutrophils and decreased the expression of ADPR,thereby inhibiting the increase of intracellular Ca2+concentration induced by TRPM2,and thus weakened the activity of PAD4 enzyme.The expression level of citrullinated protein CP also decreased with the decrease of NADPH oxidase and the blocking of TRPM2,which further confirmed the influence of ROS on TRPM2/Ca2+.In addition,neutrophil mobility,supernatant IL-1β,IL-18,expression levels and m RNA of inflammationrelated proteins NCF1 and NLRP3 in each group were analyzed,indicating that ROS and Ca2+played a role in regulating the formation of NLRP3 and inflammatory mediators.The addition of Iguratimod inhibits the expression of p NCF1 and thus affects the formation of ROS and TRPM2 activator ligand ADPR,and influences the formation of NLRP3 and inflammatory mediators through the ROS/TRPM2/Ca2+pathway and regulates the citrullination process.Conclusion:1.This study revealed that RA neutrophils had anti-inflammatory and citrullination-inducing effects,and Iguratimod had anti-inflammatory and anticitrullination-inducing effects.2.In this study,a cell model simulating RA neutrophil inflammation/citrullination was constructed,demonstrating that LPS applied to differentiated HL-60 cell lines could simulate RA neutrophil inflammation/citrullination.3.Neutrophil NADPH oxidase /ROS regulates the opening of TRPM2 by influencing the expression of TRPM2 activator ligand ADPR,thereby promoting the formation of NLRP3 and increasing the expression of inflammatory factors.Meanwhile,the activation of PAD4 leads to the enhancement of intracellular citrullination.4.The activation of TRPM2 in neutrophil granulocytes was ROS dependent,and the increased expression of Tr PM2 led to the increase of intracellular calcium ion concentration,resulting in the increased formation of NLRP3 and inflammatory factors,as well as the increased activity of PAD4 enzyme and the degree of citrullination.IGU interferes with NLRP3 inflammasome and inflammatory cytokine formation through ROS/TRPM2/Ca2+pathway by affecting the NADPH oxidase /ROS axis,and regulates intracellular citrullination induced by calcium-dependent PAD4. |
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