| Rheumatoid arthritis?RA?is a chronic,systemic autoimmune disease characterized by synovial hyperplasia,pannus formation,and progressive destruction of joints.In the early stage of RA,joint synovial inflammation caused by various reasons promotes tumor-like proliferation of synovial cells,resulting in synovial thickening,inflammatory cell infiltration,and finally pannus formation on the surface of the synovial hyperplasia.In the course of the disease,the synovial lining cells highly express matrix metalloproteinase-3?mmp-3?,which penetrates the cartilage and then erodes the bone plate on the edge of the joint,leading to the degradation of cartilage and bone,eventually leading to progressive irreversible damage and dysfunction of the joint tissues including tendon,cartilage and bone.Therefore,pannus formation is an important pathological basis for RA joint injury,deformity and loss of function.The formation of new blood vessels is a key early event of pannus formation.These new blood vessels provide nutrients and oxygen to the hypertrophic joints.There is a close relationship between synovial inflammation,synovial angiogenesis and RA pathology.Angiotensin??Ang??is one of important component of the Renin-Angiotensin System?RAS?.And it not only mediated direct physiological effects,such as vascular contraction and blood pressure regulation,but also cause some cardiovascular disease,such as high blood pressure and atherosclerosis,this may be related to it the pathological growth of blood vessels and vascular smooth muscle cell proliferation and function of necrosis,and can induce vascular inflammation,eventually leading to vascular remodeling and dysfunction.Thus,Ang?there is important relationship and inflammatory immune diseases.Ang?has two main receptor subtypes,angiotensin type 1?Ang?type 1 receptor,AT1R?and type 2 receptor?Ang?type 1 receptor,AT2R?,both are 7 times across the membrane of G protein coupled receptors.Ang?binding to AT1R has a proinflammatory role,can promote cell proliferation and so on.But Ang?binding to AT2R has anti-inflammatory effects.Research has shown that RA patients of peripheral blood mononuclear cells?PBMCs?of Ang?,AT1R and AT2R protein expression level significantly higher than healthy people.RA patients in the inflammation of the synovial tissue Ang?and AT1R level is normal synovial tissue also was increased.RA patients are more likely to suffer from vascular diseases than the general population.Studies have shown that losartan,an AT1R blocker,can reduce the level of erythrocyte sedimentation rate in patients with rheumatoid arthritis,thereby improving the inflammatory environment in the body.Vascular injury disease model mice induced by AT2R gene knockout was more severe than that induced by wild mice.However,atherosclerosis and other new vascular diseases induced by AT2R transgenic mice were alleviated compared with the wild mouse model.Therefore,AT2R has a protective effect in cardiovascular diseases.CGP42112 is an AT2R receptor selective agonist that inhibits proliferation,inflammation and remodeling,and induces vasodilation and regeneration.In the model of adjuvant arthritis in rats,injection of CGP42112 into the articular cavity of rats can effectively alleviate the inflammatory symptoms of rats with arthritis.More than a series of evidence to suggest that Ang?and its receptor play an important role on the pathogenesis of RA.Paeoniflorin-6'-O-benzosulfonate?code:CP-25?,an esterified derivative of paeoniflorin,has strong anti-inflammatory and immunomodulatory effects on rats with adjuvant arthritis and mice with collagen-induced arthritis,and alleviates synovitis of joints.CP-25 can down-regulate BAFF-TRAF2-NF-?B signaling in mice with collagen-induced arthritis and regulate the activity of spleen T cells in rats with adjuvant induced arthritis?AA?.In addition,it inhibits the function of activated B cells in humans and prevents autoimmune arthritis by modulating the immune mediators of inflammation and bone damage.Previous studies found that Ang II aggravated vascular injury in AA rats by activating AT1R/ERK1/2 signaling pathway.Whether CP-25 can inhibit the formation of RA vasospasm by the effect of Ang II and its receptor on endothelial cell function is unclear.Therefore,this study examined the effects of Ang II and its receptors on the function of HUVECs and the mechanism of action of CP-25 through Ang II receptors.Objective:This study was to observe the CP-25 of collagen induced arthritis?CIA?rats anti-angiogenesis effect,and discuss the regulation Ang?relevant mechanism and its receptor signal.Methods:Take 30 male Wistar rats,the rat tail root intradermal injection of 0.2 ml chicken?collagen type emulsion mixed with complete freund's adjuvant induced collagen arthritis rat model is established Rats with successful modeling were randomly divided into the model group and the CP-25?50mg/kg?treatment group,with an average of about 10 rats in each group,and another 10 rats with normal body weight similar to that of the normal group.Rats in the d21 and CP-25 treatment groups were given drugs by intragastric administration?0.5ml/kg?every day for two weeks.Normal group and model group rats were given the same amount of normal saline.d30,small animal ultrasound imaging technology was used to detect the changes of blood flow signal in local joints of swelling of rats in each group.On day d35,rats in each group were sacrificed,and the joints were taken for pathological examination to observe the effect of CP-25 on the formation of synovial pannus in rats.In vitro experiment,to observe the effect of CP-25?10-66 mol/L?on human umbilical vein endothelial cell?HUVEC?proliferation,migration and the influence of tube function,and separately in Ang?receptor blockers PD123319?10-66 mol/L?or Losartan?10-66 mol/L?,respectively,blocking Ang?and AT2R,the combination of AT1R research CP-25 for Ang?downstream signal.Proliferation function of HUVEC was detected by CCK 8 reagent method,migration function of HUVEC was detected by transwell method and scratch test,and tube formation function of HUVEC was detected by tube formation test.The expression of AT1R and AT2R in HUVEC was detected by laser confocal technique.The expression of Ang?receptor and GRK2,p-ERK1/2,p-I-?B?,p-P65 were detected by Western blot method.Results:1.CP-25 can significantly alleviate the pathological changes of synovial membrane of the knee joint in CIA rats.The results of ultrasound imaging showed that the blood flow signal in the tenton-tibia-femur triangle region of the rats in the d30 model group was significantly enhanced compared with that in the normal group,while the blood flow signal in the CP-25 treatment group was significantly decreased compared with that in the model group.Pathological examination showed that the synovium of the normal group was arranged in a single layer without inflammatory cell infiltration,and the cartilage and bone were intact without erosion.Compared with the normal group,the CIA model rats had obvious synovial cell proliferation,synovial thickening,inflammatory cell infiltration,increased angiogenesis,pannus formation,defects in bone and cartilage,and unclear cartilage boundary.The pathological changes of the joints in the CP-25treatment group were significantly improved,and the synovial hyperplasia,inflammatory cell infiltration,pannus formation and bone erosion were significantly improved compared with the model group.2.The effects of CP-25 combined with Ang?receptor blocker on the functions of the endotheliumTNF-??5 ng/ml?and Ang??1×10-77 mol/L?,a total of 48 h can stimulate significantly enhanced HUVEC proliferation,migration and tube function.AT1R blocker losartan?10-66 mol/L?inhibited HUVEC proliferation and migration,and reduced HUVEC tubule-forming ability.AT2R blocker PD123319?10-66 mol/L?enhanced HUVEC migration,but had no significant effect on HUVEC proliferation and tube formation.Ang?different receptor mediated the function of HUVEC.Whether blocking AT1R and AT2R,CP-25?10-66 mol/L?can effectively restrain the TNF-??5 ng/ml?and Ang?(10-7mol/L)induced HUVEC proliferation and migration.It suggests that CP-25 may play a role in HUVEC function by regulating receptor signals.3.Effect of CP-25 on AT1R/AT2R-GRK2-ERK 1/2-NF?B signaling pathwayTNF-?and Ang?combination can significantly increase the expression of AT1R and AT2R,GRK2,p-ERK,p-I?B?,p-P65.AT2R blocker PD123319?10-66 mol/L?can raise the expression of AT1R,GRK2,p-ERK,p-I?B?,p-P65.The AT1R antagonist losartan?10-66 mol/L?can down-regulate the expression of GRK2,p-ERK,p-I?B?,p-P65 and CP-25 could further down-regulate the expression of the above protein molecules.It was suggested that CP-25 could not directly affect the expression of AT1R and AT2R,and its effect on HUVEC was mainly related to the regulation of GRK2-ERK 1/2-NF?B signaling pathway.Conclusion:1.CP-25 administration by gavage could reduce the blood flow signal of swollen joints in CIA rats,reduce their arthritis index,improve joint pathology,and inhibit pannus formation.2.Ang?receptors mediated the different function of HUVEC,blocking the AT1R could inhibit HUVEC proliferation,migration and tube formation,blocking AT2R could only promote the migration of HUVEC.No matter blocking AT1R or AT2R,CP-25could effectively inhibit the proliferation,migration and tube formation of HUVEC.3.The inhibition of HUVEC by CP-25 is related to the regulation of GRK2-ERK1/2-NF?B signaling pathway. |
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