Pentraxin 3 Inhibits Fibroblast Growth Factor 2 Induced Osteoclastogenesis In Rheumatoid Arthritis

Objective:Rheumatoid Arthritis(RA)is a chronic autoimmune disease.The main pathological processes of RA include synovitis,pannus formation,cartilage destruction and bone destruction,in which the formation and differentiation of osteoclasts play an important role.RA Synovial Fibroblasts(RASFs)are key effector cells mediating synovitis and joint destruction in the course of RA disease,Studies have shown that the expression of Receptor Activator of Nuclear Factor-κB Ligand(RANKL)in RASFs increases,stimulating osteoclast generation and leading to joint erosion and bone destruction in RA patients.Basic Fibroblast Growth Factor 2(FGF2),a member of the FGFs family,exhibits biological activity by binding to FGF receptors(FGFRs)on target cells.FGF2 can stimulate osteoclast formation and promote bone resorption.Studies have shown that RASFs in RA patients overproduce FGF2,and the concentration of FGF2 in synovial fluid increases significantly,which is positively correlated with the degree of joint destruction in patients.In addition,FGF2 can stimulate peripheral blood mononuclear cells to differentiate into osteoclasts with bone resorption activity by inducing RASFs to express RANKL.Penteaxin(PTX3)is a typical acute phase reactive protein,which is highly expressed in serum,synovial tissue and synovial fluid of RA patients.PTX3 is a soluble pattern recognition receptor,which can bind to a variety of corresponding receptor ligand and participate in various biological effects such as innate immunity,inflammation and vascular remodeling.For example,PTX3 can bind FGF2 with high affinity and specificity,and prevent FGF2 from binding to the receptor,thus inhibiting FGf2-induced angiogenesis,smooth muscle cell activation and neointima formation.However,the role of PTX3 in FGF2-induced osteoclast formation has not been studied.We will investigate the role and mechanism of PTX3 in FGf2-induced RA osteoclast formation in vitro and in vivo.In the first part,the expression of FGF2,its receptors and RANKL in synovial tissue and synovial fluid was studied,and the process of FGF2 inducing the formation of RASFs osteoclasts was studied.In the second part,the effects of PTX3 on the formation of FGF2-induced RASFs osteoclasts were studied.In the third part,the effects of PTX3 on osteoclast formation in mice model of collagene-induced Arthritis(CIA)were studied.Methods:In the first part,during knee arthroscopy and knee arthroplasty,synovial tissue specimens and synovial fluid of 13 patients with confirmed active RA and 10 patients with Osteoarthritis(OA)were collected for experimental study.Factors such as FGF2 and RANKL and the major receptor of FGF2 proteinosaccharide(Heparan Sulfate)were identified by HE staining,immunohistochemical test and Westernblot test in the synovial tissues of patients with RA and OA Proteoglycan,HSPG)and Fibroblast Growth Factor Receptor 1(FGFR1)expression in synovial tissue of RA patients,then,we detected the expressions of FGF2,RANKL and other factors as well as the major receptors of FGF2,HSPG and FGFR1,in the synovial fluid of RA patients by ELISA.We cultured FGF2,anti-FGF2 and RASFs to observe the growth of RASFs cells,and detected RANKL and Cell Adhesion Molecule-1 in RASFs by quantitative fluorescence PCR and Westernblot method.The expression of osteoclast-related factors such as ICAM-1 and Osteoprotegerin(OPG),as well as pro-inflammatory cytokines such as TNF-α,IL-6 and IL-1β.Then,the levels of RANKL,ICAM-1,OPG,TNF-α,IL-6,IL-1β and other cytokines in the supernatant were detected by ELISA.In the second part,different concentrations of recombinant PTX3(r PTX3)were added into FGF2-treated RASFs cells for culture.Through the detection and analysis of various cytokines,the number of osteoclasts generated was identified by TRAP staining.125I-FGF2 cell binding assay was used to determine the effect of PTX3 on the binding of FGF2 to its receptor,so as to observe the effect of PTX3 on the formation of RASFs osteoclasts induced by endogenous and exogenous FGF2,and analyze the effect of PTX3 on the binding of FGF2 to FGFR1 and HSPG receptors on RASFs.In the third part,collagen-induced arthritis(CIA)mice were prepared by injection of type II collagen.After injecting recombinant PTX3 into CIA mice,arthritis score and Micro-CT examinations were performed on the mice to compare the changes of the injection group,CIA group and normal control group.Then,the mice were sacrificed,the synovial tissue of ankle joint was stained,and the levels of RANKL and OPG were detected by immunohistochemistry.The concentrations of RANKL and OPG were detected by serum ELISA,and the effects of PTX3 on osteoclast formation in CIA mice were observed.Results:In the first part,we found that the expressions of FGF2 and RANKL in the synovial tissue of patients with RA were significantly higher than those of patients with OA,and the levels of FGF2 and RANKL in the synovial fluid of patients with RA were also significantly higher than those of patients with OA.Immunohistochemistry and q RTPCR revealed the expression of FGFR1 and HSPG,the major receptors of FGF2,in the synovium of RA:the protein and m RNA expressions of HSPG were higher than those of OA group,while the expression of FGFR1 was not significantly different from that of OA group.We found that FGF2 could promote the growth of RASFs cells and increase the protein and m RNA expression of RANKL,ICAM-1 and RANKL in RASFs.FGF2 could lead to the increase of RANKL concentration and RANKL/OPG in RASFs culture superneant.FGF2 increased m RNA levels of TNF-α,IL-6 and IL-1β in RASFs.The addition of FGF2 resulted in an increase in the number of osteoclasts,while the addition of antiFGF2 inhibited the above changes.In the second part,different concentrations of PTX3 were added to FGF2-treated RASFs for culture,and it was found that the growth of RASFs cells in the PTX3 group and the gene and protein expressions of RANKL and ICAM-1 in the cells were significantly decreased compared with those in the FGF2 group.PTX3 decreased the concentration of RANKL and the ratio of RANKL/OPG in the superneant of RASFs culture,inhibited the m RNA expression of TNF-α,IL-6 and IL-1β in RASFs culture,and reduced the number of osteoclasts generated in TRAP staining.The above inhibitory effect was similar to that of anti-FGF2.With the increase of PTX3 concentration,the inhibitory effect was gradually enhanced.In 125I-FGF2 cell binding experiments,we found that PTX3 could inhibit the binding of FGF2 and its receptor,and the inhibitory effect was enhanced with the increase of concentration.Combined with the results of the first part,we speculated that PTX3 was highly likely to inhibit the formation of osteoclasts induced by FGF2 in RASFs by inhibiting the binding of FGF2 to HSPG.In the third part,we found that PTX3 could improve the arthritis score and bone changes of CIA mice,and r PTX3 could significantly reduce the levels of RANKL and RANKL/OPG in the local synovial membrane of the joints of CIA mice,and improve joint inflammation,cartilage and bone destruction of CIA mice.ELISA also found that r PTX3 could significantly reduce the serum RANKL level and RANKL/OPG ratio of CIA mice.Conclusions:The expressions of FGF2 and RANKL in synovium and joint fluid of RA patients are increased,and FGF2 can increase the expression levels of RANKL,ICAM-1,TNF-α,IL-6,IL-1β in RASFs,leading to the increase of osteoclast generation.PTX3 can play a similar role as anti-FGF2,inhibiting the effects of FGF2 on RASFs,and the inhibitory effect increases with the increase of PTX3 concentration within a certain concentration range.Injection of PTX3 can reduce serum RANKL concentration and RANKL/OPG ratio of mice,improve joint inflammation and bone destruction of CIA mice.PTX3 is likely to be involved in the bone destruction process of RA and has a certain protective effect on this process,which may contribute to the research on the pathogenesis of RA and provide a theoretical basis and potential therapeutic target for the treatment of RA.