G Protein-coupled Receptor Kinases 2 Regulates The Function Of Fibroblast-like Synoviocytes Through CXCR4-ERK Signaling And The Effect Of Paeoniflorin-6?-O-benzene Sulfonate(CP-25)

Rheumatoid arthritis(RA)is a chronic inflammatory autoimmune disease,characterized by synovial inflammation,destruction of bone and cartilage.Pathological process mainly inflammatory cells infiltration,synovial hyperplasia and pannus formation.Fibroblast-like synoviocytes(FLS)can release a large amount of proinflammatory molecules and substrate degradation effect,lead to immune cell infiltration and joint damage,so the abnormal FLS signal transduction is one of the important pathogenesis of RA.CXCL12,also known as stromal cell derived factor 1(SDF-1)belongs to the chemokine family and has multiple biological functions,such as stem cell mobilization,inflammatory cells infiltration and angiogenesis;Participate in all kinds of disease processes,such as cancer,AIDS,autoimmune diseases and ischemic diseases,etc.CXCL12 and its receptor CXCR4 are expressed in joint tissues and are involved in the pathological process of RA,such as inflammatory cell infiltration in joint tissues,synovial cell dysplasia and angiogenesis.The synovium,synovial fluid and serum levels of CXCL12 in RA patients were significantly higher than those in normal subjects.The pathological changes of synovium were related to CXCL12 / CXCR4 in RA patients,but the exact molecular mechanism remains unclear.CXCL12 can promote FLS proliferation,thus make synovial hyperplasia,but its mechanisms have not been reported.Paeoniflorin-6 '-O-benzene sulfonate(CP-25)as a new type of active monomers,which the main effective components of paeoniflorin(Pae)structure of esterification modification.Studies have shown that the TGP(total glucosides of paeony)and Pae has anti-inflammatory and immune regulation,can obviously inhibit arthritis rats swelling,improving joints,spleen,and mesenteric lymph nodespathological change.Pae bioavailability is low,however,causes the work slowly,therefore,the research structure of Pae modified enhance fat-soluble,new active monomer was synthesized CP – 25.Team early research has shown that CP-25 can inhibit the abnormal proliferation of FLS and down-regulate the levels of Interleukin-1 ?(IL-1?),Interleukin-6(IL-6)and Tumor necrosis factor ?(TNF-?).CP-25(25,50,100 mg·kg-1)has an exact therapeutic effect on adjuvant-induced arthritis(AA),not only can significantly reduce AA rats joint inflammation,improve the abnormal proliferation of FLS,also have immunomodulatory effect,can regulate the function of dendritic cells,improve the AA rats spleen pathology,including white pulp hyperplasia and red pulp congestion,reduce the arteries surrounding lymphatic sheath cell density,reduce lymph nodules,edge area and germinal center hyperplasia,improving synovial tissue hyperplasia,reduce pannus formation and inflammatory cells infiltration.Whether CP-25 to improve RA synovial pathology by CXCL12 / CXCR4 signaling pathway is unclear.Our previous study found that Pae inhibited the proliferation of synoviocytes in the AA and decreased the secretion of proinflammatory cytokines and mediators such as Interleukin-1(IL-1),TNF-a and Prostaglandin E2(PGE 2).Pae regulates E-prostanoid(EP)-G protein-AC-c AMP signaling pathway,up-regulates the expression of EP4 and EP2 in synoviocytes,down-regulates the expression of Gi protein,elevates the level of intracellular cyclic adenosine monophosphate(c AMP)and thus reduces the proliferation of synoviocytes.Pae can inhibit the high expression of ?-arrestin and G-protein-coupled receptor kinase 2(GRK2)in FLS of collagen-induced arthritis(CIA)rats so as to regulate the G protein coupled receptor(GPCR)coupling signals and play a role in the treatment of CIA.CP-25 can inhibit GRK2 from cytoplasmic to membrane,reduce the combination of GRK2 and EP4,which improve the function of FLS(in publish).GRK2 on the cell membrane can internalize CXCR4 by the recruitment of?-arrestin1,and GRK2 in the cytoplasm reduces ERK activation by inhibiting MEK activity to improve abnormal cell proliferation Whether CP-25 to improve the function of FLS by adjusting GRK2 and CXCR4 receptor signal is unclear.On the basis of previous studies,AA model was established successfully in this study.Using the effective dose of CP-25(mg·kg-1)to observe the synovial tissue pathological structure change.To discuss the relationship betweenthe expression of CXCL12/CXCR4 and synovial pathological changes with AA.In order to clarify CXCR4 receptor signal regulated by GRK2,and the effect of CP-25.We observe the CXCR4 receptor signal transduction pathways regulated by GRK2 and the effect of CP-25 after the CXCL12 stimulants through the transwell,immune coprecipitation and Western blot.To reveal the CP-25 provide basis for the mechanism of anti-inflammatory and immune regulation.Objective:1.To observe the expression of CXCL12 / CXCR4 in synovium of AA rats and clarify the relationship between synovium pathological changes and CXCL12 / CXCR4.2.To investigate the influence of CXCL12 on the function of FLS from normal rats and AA rats,clarify whether FLS in inflammatory conditions is more sensitive to CXCL12 than in normal conditions.3.To observe the function of FLS from AA rats stimulated by CXCL2 and the effect of CP-25,which reveal the CXCR4 receptor signaling pathways regulated by GRK2,clarify the CP-25 may by adjusting the function of FLS by GRK2 and CXCR4 receptor signal.Methods:1.AA rats were induced using complete freund's adjuvant.HE staining was used to assess the pathological changes of synovium.The expression of CXCL12 and CXCR4 in synovium was detected by ELISA,immunohistochemical and western blot.2.The proliferation of FLS was detected by CCK-8.3.The migration of FLS was detected by transwell.4.The expression of GRK2,CXCR4,ERK and p-ERK in FLS was detected by western blot.The interactions between CXCR4,GRK2 and p-ERK was detected by co-immunoprecipitation and laser scanning confocal microscopy.Results:1.Introduction of AACP-25(50?mg·kg-1)?alleviated pathological changes of synovium and decreased the expression of CXCL12 and CXCR4 in synovium of AA rats.The pathological changes of synovium and the expression of CXCL12/ CXCR4 in synovium revealed a positive correlation between the two.2.The migration of FLS in normal rats and AA rats influenced by CXCL12CXCL12(12.5,25,50,100,200 ng·m L-1)stimulation significantly promote the migration of normal rats and AA rats FLS.FLS in AA rats is higher than normal rats sensitivity to the CXCL12.3.The expression of CXCR4,GRK2,ERK and p-ERK on FLS in normal rats and AA rats induced by CXCL12The total expression of GRK2 and CXCR4,the cytoplasmic expression of CXCR4 and GRK2,the membrane expression of GRK2 and CXCR4,the expression of ERK and p-ERK of FLS in AA rats are higher than normal rats.CXCL12(100 ng·m L-1) stimulation of FLS in AA rats and normal rats with 30 minutes,their respective total expression of GRK2 and CXCR4 and ERK expression unchanged,but the total expression of CXCR4 and GRK2,ERK expression of FLS in AA rats are higher than normal rats.CXCL12 up-regulate the membrane expression of GRK2 and the cytoplasm expression of CXCR4 and the expression of p-ERK,down-regulate the cytoplasm expression of GRK2 and the membrane expression of CXCR4 in FLS with AA and normal rats.FLS in AA rats to CXCL12 stimulation sensitivity is higher than normal rats.4.The effect of CP-25 on the proliferation and migrationof FLS induced by CXCL12 in AA ratsCXCL12(50,100,200 ng·m L-1)can promote the proliferation of FLS and CP-25(10-5mol·L-1)can reduce the abnormal proliferation of FLS compared with CXCL12(50ng·m L-1).CXCL12(12.5,25,50,100,200 ng·m L-1)can promote the migration of FLS and CP-25(10-8,10-7,10-6,10-5mol·L-1)can reduce the abnormal migration of FLS compared with CXCL12(50 ng·m L-1).5.The effect of CP-25 on the expression of CXCR4,GRK2,ERK and p-ERK on FLS induced by CXCL12 in AA ratsCXCL12(100 ng·m L-1)stimulation of FLS in AA rats with 30 minutes,the total expression of GRK2 and CXCR4 and ERK expression unchanged.CXCL12 up-regulate the membrane expression of GRK2 and the cytoplasm expression of CXCR4 and the expression of p-ERK,down-regulate the cytoplasm expression of GRK2 and the membrane expression of CXCR4 in FLS with AA rats.CP-25(10-7,10-5mol·L-1)down-regulate the membrane expression of GRK2 and the cytoplasm expression of CXCR4 and the expression of p-ERK,up-regulate the cytoplasm expression of GRK2 and the membrane expression of CXCR4 in FLS with AA rats.CXCL12(100 ng·m L-1)stimulation of FLS in AA rats with 30 minutes,up-regulate the interaction between GRK2 and CXCR4 and down-regulate the interaction between GRK2 and p – ERK.CP-25 can down-regulate the interaction between GRK2 and CXCR4 and up-regulate the interaction between GRK2 and p-ERK.Conclusion:1.Increased expression of CXCL12 and its receptor CXCR4 may be associated with the pathological changes of synovium in AA rats,which play an important role in the pathogenesis of RA.CP-25 had obvious therapeutic effect on AA rats and its mechanism may be related to inhibition the expression of CXCL12 and CXCR4 in the synovium.2.The migration of FLS in AA rats obviously than normal rats induced by CXCL12.CXCL12 can promote FLS proliferation and migration,this effect may be through the promotion of cytoplasmic GRK2 transposition to the cell membrane,abate GRK2 in the cytoplasm of ERK inhibition,leading to increased ERK activation.3.CP-25 inhibiting GRK2 transfer from cytoplasm to membrane,restore the cytoplasm of GRK2 inhibit the activation of ERK,which the mechanism of its down-regulate the proliferation and migrationof FLS.