The Effect Of Platelet Microparticles On RANKL/OPG System In Rheumatoid Arthritis Fibroblast-like Synoviocytes

ObjeciveRheumatoid arthritis(RA)is a chronic autoimmune disease which is mainly characterized by joint synovial hyperplasia,inflammatory cell infiltration,pannus formation and cartilage and bone destruction and the pathogenesis of RA is not yet fully clear.With the deepening of the research,rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)is considered to be the key effector in the pathogenesis and development of RA.Activated RA-FLS has the properties of tumor cell,such as abnormal proliferation,apoptosis inhibition and enhanced ability of migration and invasion,and plays an important role in RA synovial hyperplasia,angiogenesis and cartilage and bone erosion.Platelet microparticles(PMPs)are produced during the process of platelet activation.It has been reported that the number of PMPs was increased in peripheral blood and joint fluid of RA patients and PMPs could promote the inflammatory response of RA,suggesting that PMPs may be closely related to the occurrence and development of RA.Receptor activator of NF-?B ligand(RANKL)/Receptor activator of NF-?B(RANK)/Osteoprotegerin(OPG)system palys a regulatory role in bone destruction and bone remodeling,and the imbalance of RANKL/OPG ratio is an important factor in bone destruction of RA.In this research,we studied the effects of PMPs on the expression of RANKL and OPG in RA-FLS and investigated the regulation of PMPs on RANKL/OPG system and possible mechanism,which would provide a novel target for the diagnosis and treatment of RA.Methods1.PMPs were extracted from activated platelet rich plasma and the purity was measured with CD41 antibody by flow cytometry.The concentration of PMPs was determined by BCA method.2.MH7A cell strain was labeled with CD90 and CD55 antibodies,and then identified by flow cytometry.3.The effect of PMPs on the proliferation of RA-FLS was explored by MTT assay,cell counting assay and crystal violet staining assay.4.The effect of PMPs on the apoptosis of RA-FLS was detected by flow cytometry after PI staining and Annexin V-FITC/PI double staining,respectively5.High throughput gene chip was used to analyze gene expression profile of RA-FLS after incubation with different concentrations of PMPs.6.RT-qPCR was used to verify the expression of RANKL and OPG of RA-FLS after treatment with PMPs in the mRNA level.The expression and 'secretion of RANKL and OPG were detected by Western Blot and ELISA,respectively.7.Western Blot was used to explore the effects of PMPs on the expression of RANKL and OPG in RA-FLS and the possible mechanism possible mechanism combined use of signal pathway inhibitors.Results1.The purity of PMPs detected by flow cytometry was 93.4± 1.46%and could be used for subsequent experiments.The concentration of PMPs measured by BCA method was 800 ?g/ml.2.The percentage of MH7A expressing CD55+/CD90+ was 95.6 ± 1.36%,indicating the cell could be used for later experiments.3.The results from MTT assay,cell counting assay and crystal violet staining assay showed that PMPs could significantly promote the proliferation of RA-FLS.4.The results from PI staining demonstrated that there was no obvious Sub-G1 peak in RA-FLS with different concentrations of PMPs.The results from Annexin V-FITC/PI double staining showed that there was no statistical significance in the apoptosis rate of RA-FLS after treatment with PMPs when compared with the control group(0 ?g/ml PMPs).All the results indicated that PMPs had no obvious effect on the apoptosis of RA-FLS.5.High throughput gene chip analysis showed that there have been significantly alteration in 298 gene expression of the group of 25 ?g/ml PMPs,164 gene expression in the group of 50 ?g/ml PMPs and 39 gene expression in both group.6.Results from the gene chip analysis showed that there have been up-regulation of RANKL and down-regulation of OPG in the group of 25 and 50 ?g/ml PMPs when the control group.The results from RT-qPCR which was used to verify the results of gene chip showed that the expression of RANKL was increased and the expression of OPG was decreased in RA-FLS after treatment with PMP(P<0.05).The results from Western Blot and ELISA showed that PMPs promoted the expression and secretion of RANKL,inhibited the expression and secretion of OPG and increased the ratio of RANKL/OPG(P<0.05).7.The results of Western Blot showed that:A.When combined use of NF-?B inhibitor to RA-FLS after treatment with PMPs,the expression of RANKL down-regulated and the expression of OPG up-regulated,the RANKL/OPG ratio was increased remarkably,indicating that PMPs may regulate the RANKL/OPG ratio through NF-?B pathway.B.After adding SB225002(CXCR2 inhibitor)to RA-FLS after treatment with PMPs,the expression of RANKL was increased,the expression of OPG decreased,and RANKL/OPG ratio was significantly lower than that in PMPs group,suggesting that PMPs may activate NF-?B through CXCR2 and then regulate the RANKL/OPG ratio.C.Compared with the control group,the expression of p-Erk in PMPs group increased and the expression of Erk was not significantly changed.After addition of CXCR2 inhibitor SB225002,the expression of p-Erk was significantly lower than that of PMPs group,and p-Erk/Erk ratio decreased,while Akt,JNK,p3 8 and their phosphorylation levels were not significantly changed,suggesting that PMPs might activate the phosphorylation of Erk through chemokine-CXCR2.D.After adding PD98059(Erk inhibitor)to RA-FLS incubated with PMPs,the expression of p-Erk,p-I?B and p-NF-KB decreased significantly than that of PMPs group,and Erk,I?B and NF-?B were not significantly changed,indicating that PMPs might activate NF-?B through Erk phosphorylation.All the results demonstrated that PMPs might activate NF-?B pathway mediated by Erk phosphorylation though CXCR2,and then affect the expression of RANKL and OPG in RA-FLS,regulate the RANKL/OPG ratio,and play a role in bone destruction.ConclusionPMPs could promote the proliferation of RA-FLS and might affect the expression of RANKL and OPG through activating CXCR2/Erk/NF-KB signaling pathway,regulate the ratio of RANKL/OPG,then promote the bone destruction of RA and play an important role in the pathogenesis and development of RA.