| INTRODUCTION Rheumatoid arthritis(RA)is a chronic inflammatory autoimmune disease,its typical pathological features includes: inflammation of arthritis,conformation of pannus,synovial hyperplasia and the damage of cartilage and bone.Fibroblast-like synoviocytes(FLS)are the mainly components of RA synovial tissue,accounting for about 70%,and over-activated FLS has the ability of tumor-like proliferation and migration.Aberrant migration of FLS to the cartilage can cause the formation of pannus and damage of joint.CXCL12 is a chemokine that increased in the synovial of RA patients.CXCR4 is a GPCR that binds to CXCL12 specifically,and when abnormal changes appears in the signal axis of CXCL12/CXCR4 can lead to excessive migration of FLS.Paeoniflorin-6 ’-O-benzene sulfonate(code:CP-25),is a new active compound that was obtained from the main ingredient(paeoniflorin,Pae)of total glucosides of paeony(TGP)of my group,and compared with Pae,it has well fat solubility and improved bioavailability.According to the previous studies of my research group, CP-25 can significantly alleviated the index of arthritis,degree of joint swelling,count of joint swelling,moderated the pathology of joint and spleen.At the level of cells,CP-25 can inhibit the proliferation and migration abilities of FLS,inhibit the ability of FLS and macrophages as well as T cells in secreting inflammatory cytokines.GRK2 is a Ser/Thr protein kinase,its main role is to participate in the desensitization and internalization of GPCR.Furthermore,it also can participate downstream signaling pathway(PI3K/AKT,ERK)via interaction with other molecules.Changes of GRK2 levels in immune cells play an important role in the process of inflammatory immune response,and in CIA mouse model,the protein expression level of GRK2 is increased,which promotes the development of arthritis.The previous study showed that CP-25 regulated ERK1/2 signaling pathway through inhibiting GRK2 translocation from cytoplasm to cell membrane,thus reducing angiogenesis in HUVECs.CP-25 increased the membrane expression of β2-AR and decreased the membrane expression of GRK2 in synoviocytes.Previous study showed that Pae could reduce the protein expression of PI3K/AKT signaling pathway in collagen-induced arthritis models,thus play a therapeutic role in CIA rats.PI3K/AKT signaling pathway is a classical signaling pathway associated with cell proliferation,migration,apoptosis and angiogenesis.Many studies have shown that over-activation of PI3K/AKT signaling pathway is related to the migration process of cancer cells.In addition,when GPCR combined with corresponding ligand,it can lead to the ac tivation of G protein,cause the dissociation of Gα and Gβγ subunit,dissociated Gβγ subunit can recruit molecules to the cell membrane,such as GRK2,PI3 K,and activate PI3 K on the membrane.Therefore,we hypothesized whether CP-25 regulates the excessive migration of FLS in RA is related to GRK2 and its interaction molecule,such as PI3K/AKT signal pathway? We choosed synovial tissue of RA and OA patients,as well as MH7 A cell lines as our research object,and expounded the molecular mechanism of FLS over-migration in RA from the clinical samples and cell lines levels.Transwell was used to detect whether CP-25 can inhibit the excessive migration of FLS in RA,Western blot,Co-IP and LSCM were used to further detect whether its molecular mechanism is related to GRK2 and its interaction molecule,such as PI3K/AKT signal pathway.OBJECTIVE 1.To identify whether over-activation of PI3K/AKT signaling pathway in synovial tissue of RA patients is involved in highly expression of CXCR4.2.To identify whether CP-25 participate in the migration function of FLS is mediated by PI3K/AKT signal pathway.3.To identify the specific molecular mechanism of CP-25 in regulating PI3K/AKT signaling pathway.METHODS 1.Synovial tissues of RA and OA patients were collected,and the proteins expression of CXCR4,GRK2,Gβ,p110β,p85,p-p85,AKT,p-AKT in synovial tissue of RA and OA patients were detected by Western blot and Immunohistochemistry;2.The migration of MH7 A stimulated by CXCL12 and the role of CP-25 on it were detected by transwell;3.The proteins expression of GRK2,Gβ,p110β,p85,p-p85,AKT and p-AKT stimulated by CXCL12 and the role of CP-25 on it were detected by Western blot;4.The membrane and cytoplasm protein expression of GRK2,Gβ and PI3Kβ(p110β)stimulated by CXCL12 and the role of CP-25 on it were detected by Western blot;5.The co-expression of GRK2 and Gβ,PI3Kβ(p110β)and Gβ stimulated by CXCL12 and the role of CP-25 were detected by Co-IP and LSCM.RESULTS 1.Changes of CXCR4,GRK2,Gβ,p110β,p85,p-p85,AKT and p-AKT protein expression in synovial tissues of RA and OA patients 8 synovial tissue of RA patients and 9 of O A patients were collected.Compared with OA patients,the expression of CXCR4,GRK2,Gβ,p-p85 and p-AKT proteins in RA patients was elevated,indicating over-activation of PI3K/AKT signaling pathways in RA patients.2.The correlation analysis between CXCR4 receptors and p-p85,p-AKT protein expression in synovial tissues of RA and OA patients Compared with the synovial tissues of O A patients,the exp ression of CXCR4 receptor is increased in the synovial tissues of RA patients,and there exist a positive correlation between the expression of C XCR4 and over-expression of p-p85,p-AKT.The results indicated that highly expression of CXC R4 is associated with over-activation of PI3K/AKT signaling pathway.3.The effect of CXCL12 on the migration of MH7 A and the role of CP-25 Compared with control group,after stimulation with 100ng/ml CXC L12 for 30 min,the migration of MH7 A is increased.Compared with the 100ng/ml C XCL12 group,CP-25 with concentrations of 0.1μM and 1μM reduced the migration of MH7A;Gallein reduced migration of MH7 A at concentrations of 10μM and 20μM compared with the 100ng/ml CXCL12 group.4.The effect of CXCL12 on the protein expression of GRK2,Gβ,p110β,p85,p-p85,AKT and p-AKT in MH7 A and the role of CP-25 Compared with control group,after stimulation with 100ng/ml CXC L12 for 30 min,the protein expression of p-p85 and p-AKT was increased in MH7 A,but the protein expression of GRK2,Gβ,p110β,p85 and AKT protein showed no significant change.Compared with the 100ng/ml CXC L12 group,CP-25 with concentrations of 0.1μM and 1μM,the protein expression of p-p85 and p-AKT were decreased in MH7 A,but the protein expression of GRK2,Gβ,p110β,p85 and AKT protein showed no significant change.Compared with 100ng/ml CXCL12 group,gallein decreased the protein expression of p-p85 and p-AKT in MH7 A at concentrations of 10μM and 20μM,but the protein expression of GRK2,Gβ,p110β,p85 and AKT protein showed no significant change.5.Effects of CXCL12 on the membrane and cytoplasm expression of GRK2,Gβ,PI3 K and role of CP-25 in MH7 A.Compared with control group,after stimulation of 100ng/ml CXC L12 for 30 min,the membrane protein expression of GRK2,Gβ and PI3 K were increased in MH7 A,while the cytoplasm protein expression of GRK2,Gβ and PI3 K were decreased.Compared with 100ng/ml CXC L12 group,CP-25 of 1μM decreased the membrane expression of GRK2,Gβ and PI3 K in MH7 A,and increased the cytoplasm expression of GRK2,Gβ and PI3 K.6.The effect of CXCL12 on the co-expression of GRK2 and Gβ、Gβ and PI3 K in MH7 A and the role of CP-25 Compared with control group,after stimulation with 100ng/ml CXC L12 for 30 min,the co-expression of GRK2 and Gβ,Gβ and PI3 K in MH7 A were increased.Compared with the 100ng/ml CXC L12 group,CP-25 of 1μM reduced the co-expression of GRK2 and Gβ,Gβ and PI3 K in MH7 A.CONCLUSION 1.In the synovial tissues of RA patients,the protein expression of CXCR4,GRK2,Gβ,p-p85 and p-AKT is increased,and there exist a positive correlation between expression of CXCR4 receptor and activation of PI3K/AKT signaling pathway.2.CP-25 inhibited the CXCL12 mediated excessive migration of MH7 A by down-regulating PI3K/AKT signaling pathway.3.CP-25 down-regulates PI3K/AKT signaling pathway in MH7 A may be related to inhibition of GRK2-Gβγ membrane translocation,therefore indirectly reduced membrane expression and activation of PI3 K,down-regulated overactivation of AKT. |
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