| 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.Synovial neovascularization and pannus formation are the typical characteristic in RA.Intra-articular macrophages,synovial fibroblasts and endothelial cells secrete a variety of proangiogenic factors,such as vascular endothelial growth factor,hypoxia-inducible factor,chemokines and pro-inflammatory cytokines.These proangiogenic factors can maintain synovial and intra-articular inflammation through neovascularization.CXCL12 belongs to the chemokine family and has multiple biological functions,such as chemotaxis to lymphocyte and participates in the occurrence and development of many diseases,such as AIDS,cancer,autoimmune diseases and so on.CXCL12/CXCR4 axis is highly expressed in synovial cells and endothelial cells in RA and participate in the pathological process of RA,such as inflammatory cell infiltration in joint tissue,synovial cell hyperplasia and pannus formation.The pannus formation is one of the reasons of synovial hyperplasia and inflammation in RA patients.CXCL12/CXCR4 axis is involved in vascular remodeling and angiogenesis in RA.It was found that the vascular remodeling was disordered,the new endothelial cells form was abnormal and angiogenesis was also suppressed in rat arthritis model after applied the AMD3100 which was the CXCL12/CXCR4 axis blockers.CXCL12 can regulate the ERK1/2 activation through G protein-coupled receptor signaling pathway,which is an important mechanism in the process of angiogenesis.In endothelial cells,GRK2 negatively regulates the CXCR4 receptor,which can mediate the CXCR4 desensitization and endocytosis.Paeoniflorin-6’-O-benzene sulfonate（CP-25）,as a new type of active monomers is a novel ester derivative of paeoniflorin（Pae）.Our previous study revealed that the oral and venous pharmacokinetic parameters of CP-25 were increased compared with Pae and that CP-25(25,50,100 mg·kg-1)had been demonstrated to have therapeutic effects on adjuvant-induced arthritis,which can significantly reduce the joint inflammation and inhibit synovium hyperplasia in AA rats.However,its effect on angiogenesis and pannus of AA joint is still unclear.The article was aimed to observe the proliferation of articular blood vessels,the function of HUVEC cells and the effects of CP-25 in AA model,and in order to illuminate the role of CXCL12/CXCR4 axis signal transduction in the pathological of RA.To reveal the CP-25 mechanism of anti-inflammatory and immune regulation.Objective:1.To observe the therapeutic effects of CP-25 on AA rats and the expression of CXCL12/CXCR4 axis in synovial tissue,serum and spleen tissue of AA rats,and to clarify the role of CXCL12/CXCR4 axis in AA rats.2.To observe the expression of CXCL12/CXCR4 axis of synovial vessels in AA rats and clarify the relationship between CXCL12/CXCR4 axis and angiogenesis.3.To observe the function of HUVECs stimulated by CXCL12 and the effects of CP-25 and clarify the expression of GRK2,CXCR4 and ERK1/2 which reveal the role of GRK2 in improving endothelial cell function and by regulating CXCR4-ERK signaling pathway.Methods:1.AA was initiated in the SD rats via intradermal immunization in the right hind metatarsal footpad with the heat-killed Mycobacterium butyricum in liquid paraffin.The rats were randomly divided into four groups: Normal（no immunization）,AA,CP-25(50 mg·kg-1)and MTX(0.5 mg·kg-1).The rats were given intragastric administration on the 17 th day and executed on the 30 th day.Multiple arthritis index,joint swelling index,body weight,histopathology,synovitis inflammation score and pannus score were recorded.2.The pathological of joint synovium and spleen in AA rats were evaluated by HE staining,the synovial vascular proliferation was evaluated by immunofluorescence,the expression of CXCL12 in synovium,spleen and serum was detected by immunohistochemistry and ELISA,the expression of CXCR4 in synovium and spleen was detected by Western blot.3.The proliferation of HUVECs was detected by CCK-8,the migration of HUVECs was detected by transwell,the tube-forming ability of HUVECs by tube formation assay.4.The expression of total,membrane,cytoplasm GRK2,CXCR4 and ERK,p-ERK in HUVECs was detected by western blot.The co-expression of GRK2 with CXCR4 and,p-ERK was analyzed by co-immunoprecipitationResults:1.CP-25 attenuates the clinical signs and the histopathology of spleen,ankle joint of AA,inhibits the proliferation of synovial cells and angiogenesis.CP-25(50?mg·kg-1)significantly alleviated the arthritis symptoms of AA,including global assessment and joint swelling index.CP-25(50?mg·kg-1)also improved the histopathology of spleen in AA,including reducing the number of lymphocytes around the arteries.Meanwhile,the histopathology of ankle joints suggested that CP-25 significantly reduced synovial tissue proliferation,inflammatory cell infiltration,synovial tissue inflammatory infiltration score and pannus score.2.The effects of CP-25 on the expression of CXCL12/CXCR in synovium,spleen and the expression of CXCL12 in serum of AA.The expression of CXCL12/CXCR4 in the synovium and spleen in the AA model group was increased compared with those in the normal group.CP-25 could down-regulate the expression of CXCL12/CXCR4 in synovium and spleen.Moreover,there was a positive correlation between the pathological changes indicated by the pannus score and the expression of CXCL12/CXCR4.3.Effects of CP-25 on HUVECs proliferation,migration and tube formation induced by CXCL12 in vitro.The results demonstrated that stimulation with high CXCL12(50,100 and 200ng·m L-1)significantly promoted HUVEC proliferation.CP-25(10-5,10-6mol·L-1)could significantly reduce the abnormal proliferation of HUVEC induced by CXCL12(50 ng·m L-1).Additionally,12.5,25,50,100 and 200 ng·m L-1CXCL12 could significantly promoted the migration of HUVECs and reached the peak at 24 hours,and CP-25(10-5,10-6,10-7,10-8,10-9mol·L-1)could significantly reduce the abnormal migration of HUVEC induced by CXCL12(50 ng·m L-1),the stimulation of CXCL12(12.5,25,50,100,200 ng.m L-1)significantly increased the ability of HUVECs to form tubes,while CP-25(10-5,10-6,10-7,10-8,10-9mol·L-1)significantly inhibited the ability of HUVEC to form tubes induced by CXCL12(50 ng·m L-1).4.Effects of CP-25 on the expression of GRK2,CXCR4,ERK and p-ERK in HUVECs induced by CXCL12 in vitro.The total and membranal expression of the GRK2 were significantly upregulated,the cytoplasmic expression of GRK2 was significantly downregulated following CXCL12(100 ng·m L-1)stimulation of HUVECs.The total expression of CXCR4 was not significantly altered by CXCL12 or treatment with CP-25.The membranal expression of the CXCR4 was significantly downregulated,the cytoplasmic expression of CXCR4 was significantly upregulated following CXCL12(100 ng·m L-1) stimulation of HUVECs.While CP-25(10-5,10-6mol·L-1)could regulate the abnormal expression of GRK2 and CXCR4.There was no difference in the expression of ERK induced by CXCL12(100 ng·m L-1)and CP-25(10-5、10-6、10-7mol·L-1).However,the expression of p-ERK in CXCL12-treated cells was significantly upregulated in HUVECs induced by CXCL12(100 ng·m L-1),and which was downregulated by CP-25(10-5、10-6、10-7mol·L-1).The co-expression of GRK2 and CXCR4 was upregulated and the co-expression of GRK2 and p-ERK in HUVECs induced by CXCL12(100 ng·m L-1).CP-25(10-6mol·L-1)could antagonize the effect of CXCL12.Conclusion:1.CP-25 has therapeutic effect on AA rats which can reduce the global assessment and the paw swelling of AA rats.2.The high expression of CXCL12 and its receptor CXCR4 is related to the inflammatory lesions of synovium and angiogenesis in AA rats.3.CXCL12 enhanced HUVECs proliferation,migration and tube formation,while CP-25 could regulate the abnormal variation.4.The therapeutic effect of CP-25 on AA was related to the inhibition of angiogenesis.CP-25 inhibiting GRK2 transfer from cytoplasm to membrane,restore the cytoplasm of GRK2 inhibit the activation of ERK,which was the mechanism of its downregulate the proliferation,migration and tube-forming ability of HUVECs. |
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