Effect Of Paroxetine On The Activation And The Infiltration Of T Cells In Synovial Tissues Of Rats With Collagen-induced Arthritis

Rheumatoid arthritis(RA)is a common autoimmune disease,the basic pathological changes of tissue deterioration,exudation and hyperplasia,typical acute inflammation manifested as red,swollen,hot pain and dysfunction.Cells involved in the inflammatory response can be called inflammatory cells,in which lymphocytes and macrophages are the central cells of immunoinflammatory.Early diagnosis and treatment is the key to prevention and treatment of joint injury.The use of disease-modifying anti-rheumatic drugs(DMARDs)or combination of biological agents is the basic strategy of RA treatment,but these drugs have obvious adverse effects,and the efficiency is not high,so the further study of the pathogenesis of RA,looking for new therapeutic targets and the development of new effective drugs are the main direction of RA research.There are a variety of immune cells including T cells,B cells and macrophages play its role in migrating chemokines to synovial tissue on the early stage of RA is the initiation of RA arthritis factors.Chemokine receptor is G protein coupled receptor(GPCR),whose function is regulated by G protein coupled receptor kinase(GRKs).It is unclear whether the regulation of GRKs function can reduce the migration of immune cells to synovial tissue.Paroxetine is a selective GRK2 inhibitor,which has not been reported to inhibit arthritis by inhibiting GRK2 effects on immune cell activation and infiltration in synovial tissue.Objective: To investigate the effect of GRK2 on immune cell activation and infiltration in synovial tissue of collagen related arthritis(CIA)and the molecular mechanism of paroxetine in treating CIA.Methods: The CIA model were established by Wistar rats.There was began to appear arthritis around the day of d14,and the model group rats were randomly divided into solvent control group(CIA-Veh),paroxetine(15 mg/kg/d)(CIA-Par)and MTX(0.5 mg/kg/3d)(CIA-MTX)according to the arthritis index,intragastric administration paroxetine and MTX for 15 days,and the whole indexes were evaluated every 3 days.The rats were sacrificed at the end of the administration,we would dectect ankle pathologic examination and grading;calculating the thymus and spleen index;CCK measures vitality of spleen T cells;the levels of tumor necrosis factor(TNF-?),interleukin-1?(IL-1?)and chemokine C-X3-C motif ligand 1(CX3CL1)in serum and synovial homogenate were measured by ELISA;the content of T cells,B cells and macrophages in synovial tissue and the percentage changes of T cells subgroup in peripheral blood were tested by flow cytometry;immunohistochemical was used to observe the infiltration of T cells in synovial tissue;Transwell method to detect migrated ability of T cell;the system of lipofectamine 3000 transfect p IRES-EGFP-GRK2 and p IRES-EGFP plasmid to spleen T cells;Western blot and immunofluorescence test the expression of GRK2 in T cells that contain plasmid;Western blot method to detect the different group of spleen T cells with its molecule expression and phosphorylation of GRK2 and MAPK pathways.Results:1.Effect of paroxetine on overall index of CIA rats.In the early stage of CIA rats(about 14 days after the first inflammation),paroxetine and MTX treatment group rats which systemic performance score,paw swelling,arthritis index and joint swelling were significantly decreased compared with the CIA model group,and weight were obviously get well.2.Effect of paroxetine on pathological changes of CIA ankle joint in ratsAnkle pathological examination found that the CIA model rats with a significantly hyperplasia synovium,more immune cells infiltration,and obviously pannus formationand blood vessels expansion.It had a significantly improvement of ankle pathological in paroxetine and MTX-treated rats.3.Effect of paroxetine on thymus and spleen index in CIA ratsThe thymus and spleen of CIA rats were more sweller than normal rats,and the index was significantly increased.Paroxetine and MTX group with a significant reduced in thymus and spleen index.4.Effect of paroxetine on cytokines and chemokines levels in serum and synovial tissue of CIA ratsThe levels of TNF-?,IL-1? and CX3CL1 in serum and synovial homogenate of CIA rats were significantly increased,and paroxetine and MTX administration significantly reduced the above-mentioned factor levels in CIA rat serum and synovium 5.Effect of paroxetine on the infiltration of immune cells in synovial tissueA large number of CD4+ helper T cells(Th),CD8+ cytotoxic T cells(Tc),CD45R+ B cells,CD11b+ macrophages were detected in the single cell digestive fluid of synovial tissue of CIA rats.The treatment of paroxetine and MTX significantly reduced the infiltration of Th and Tc cells in synovium tissue.MTX can inhibit the infiltration of B cells and macrophages in synovial tissue,and paroxetine has no significant effect on the migration of B cells and macrophages.Immunohistochemical results showed that there were more T cell infiltration in the synovial membrane of CIA rats.Paroxetine and MTX treatment could reduce the number of T cells in synovial tissue in different degrees.6.Effect of paroxetine on T lymphocyte viability and activationCompare with normal rats,CIA rats with a significantly increase of CD3+CD4+ helper T cells(helper T,Th),CD4+CD25+Foxp3-activated Th cells(Thact),CD4+CD44+ effector Th cells(Theff),CD8+CD44+ Tc cells(effctor cytotoxic T,Tceff)in peripheral blood,CD4+CD62L+ na?ve Th cells(Thnaive),CD4+CD25+Foxp3+ regulatory T cells(Treg),CD8+CD62L+ na?ve cytotoxic T cells(Tcnaive)were significantly deceased,and the ratio of Th/Tc,Theff/Thnaive,Tceff/Tcnaive increased,and the treatment of paroxetineand MTX could significantly recover the balance of T cell subsets.CCK detection found that the vitality of spleen T cells in CIA rats were obvious enhanced,and paroxetine and MTX can effectively reduce vitality of spleen T cells.7.The role of GRK2 in T cell migration and the effect of paroxetineWestern blot analysis showed that the expression of GRK2 in spleen T cells was significantly increased after d14,and reached the peak on the day of d28 and the remission period had a decrease tendency.In vivo,paroxetine significantly reduced the expression of GRK2 in spleen T cells.The purified spleen T cells were stimulated with CX3CL1 in vitro,and induced their migration.At the same time,paroxetine and MTX were given at different concentrations and the detection found that paroxetine and MTX in vitro significantly inhibited T cell migration.The high expression of GRK2 in T cell can promote migration,the inhibitory effect of paroxetine on GRK2 was attenuated,suggesting that paroxetine affecting the GRK2 activity to prevent T cell migration.8.Effects of paroxetine on MAPK signaling pathway in spleen T cellsSpleen T cells of CIA with a significantly increased in activating of p38 MAPK and ERK,and JNK activation is not obvious,paroxetine can significantly inhibit the phosphorylation of ERK,and MAPK signal pathway activity was not significantly affected by MTX.Conclusions:1.Paroxetine can effect improvement CIA rats.2.Paroxetine reduces the infiltration of T cells in synovial tissue of CIA rats and inhibits the activation and differentiation of T cells.3.Paroxetine reduces the infiltration of T cells in synovial tissue by inhibiting GRK2 expression and ERK phosphorylation.