| Rheumatoid arthritis(RA)is a chronic,refractory autoimmune disease.Its main pathological features are inflammatory hyperplasia of the synovial membrane,neovascularization and vasospasm,which eventually lead to the destruction of cartilage and bone.So far,the exact etiology and pathogenesis of RA is unclear,and many studies have shown that it is associated with many complex factors such as infection and heredity.Recent studies have found that the inflammatory microenvironment is closely related to the occurrence and development of RA.Many studies have shown that RA,regardless of the cause,is often associated with microenvironmental imbalances in the synovial membrane.As major participant in the microenvironment,Changes of fibroblast-like synoviocytes(FLS)and vascular endothelial cells(VEC)are closely related to microenvironmental imbalances in the synovial membrane of the joint.Inflammation plays an important role in the occurrence and progression of RA.The persistence of arthritic microenvironment can promote the occurrence and development of RA.In the inflammatory microenvironment of chronic inflammation,a large number of inflammatory factors such as sphingosine 1-phosphate(S1P),tumor necrosis factor-alpha(TNF-?),interleukin-1(IL-1)and so on,which play an important role in the occurrence and development of RA.FLS and VEC are important components of synovial tissue.VEC provides cytokines,oxygen and nutrients for FLS,while VEC can secrete a large amount of S1 P and activate the S1 P downstream signal pathway in FLS,which is one of the important factors that affect RA.Studies have shown that S1 P binds to sphingosine-1-phosphate receptors(S1PRs)and participates in the secretion,proliferation and apoptosis of inflammatory cytokines in FLS,and then regulates the development of RA.Based on the research status of VEC and FLS in the inflammatory microenvironment and its potential significance in the development of RA,this study intends to establish an inflammation injury model of human umbilical vein endothelial cells(HUVEC)induced by TNF-?.Detection of S1 P content in HUVEC,and HUVEC conditioned medium was used to culture rheumatoid arthritis fibroblasts(RASFs)to explore whether S1 P is the key molecule to the effect of VEC on FLS.To elucidate the possible molecular mechanisms by which S1 P participates in the role of VEC in FLS.Furthermore,we further explore the possible target of GE intervention in HUVEC,the cascade of S1 P signaling pathway,and anti-inflammatory immune effect.It provides new targets and strategies for the prevention and treatment of RA by anti-inflammatory and immunological active ingredients of traditional Chinese medicine,and also provides more experimental basis for the research and application of active ingredients of traditional Chinese medicine.1 OBJECTIVETo observe the expression changes of S1 P in TNF-?-induced HUVEC inflammatory model,the effect of HUVEC conditioned medium on the proliferation of RASFs and the secretion of cytokines,and to explore the molecular mechanism of VEC on RASFs through S1 P signal transduction.To observe whether GE administration can decrease the expression of S1 P in HUVEC,inhibit the S1 P downstream Ras-Erk1/2 signaling pathway in RASFs,and elucidate the target of GE and mechanism of anti-inflammatory immunity.2 METHODHUVEC was cultured in vitro,and establishment of TNF-?-induced HUVEC inflammatory injury model.ELISA was used to detect the secretion of S1 P in HUVEC.To investigate how S1 P is produced in HUVEC and the target of GE,the expression of Erk1/2 and SphK1 genes and protein in HUVEC were detected by RT-qPCR and Western blotting.In order to investigate the molecular mechanism of S1 P signal transduction in the role of VEC on RASFs,different groups of HUVEC conditioned medium were used to culture RASFs.CCK-8 and ELISA were used to detect the proliferation activity and cytokine secretion of HUVEC conditioned medium on RASFs.RT-qPCR and Western blotting was used to detect the effect of on the expression of key genes and key proteins in Ras-Erk1/2 signaling pathway in RASFs.3 RESULTS 3.1 The effect of GE on the secretion of S1 P in HUVECThe changes of intracellular and extracellular S1 P content in different groups were consistent.Compared with the control group,the S1 P content in TNF-? group was significantly increased(P<0.01).The S1 P content in GE group was decreased,and the 100 ?g/ mL GE down-regulation was the most significant(P<0.01).FTY-720(1?M)and SCH772984(1?M)also reduced the content of S1P(P<0.01).3.2 The effect of GE on the membrane and cytoplasmic expression of P-SphK1 protein in HUVECThe expression of P-SphK1 protein in cytoplasm and membrane in TNF-? group was significantly higher than that of the control group(P<0.01),suggesting that TNF-? induced P-SphK1 protein transfer to the cell membrane and promoted the formation of S1 P.Compared with TNF-? group,the expression of P-SphK1 protein in cytoplasm and membrane were significantly decreased after treatment with different concentrations of GE,FTY-720(1?M)and SCH772984(1?M)(P<0.01).3.3 Effects of GE on co-expression of P-Erk1/2 and SphK1 in HUVECUnder normal conditions,P-Erk1/2 was co-expressed with SphK1 in HUVEC.the co-expression of P-Erk1/2 and SphK1 was significantly increased in TNF-? group,compared with the control group(P<0.01).Compared with TNF-? group,the coexpression of P-Erk1/2 and SphK1 in GE group was decreased(P<0.01),which was consistent with the FTY-720 and SCH772984 groups.3.4 The effect of GE on Erk1/2 and SphK1 gene expression induced by TNF-? in HUVECThe expression of Erk1/2mRNA and SphK1 mRNA in HUVEC of TNF-? group was significantly higher than that of normal group(P<0.01).After GE treatment,the Erk1/2mRNA and SphK1 mRNA in middle and high dose groups of GE were significantly decreased(P< 0.01),and in a dose-dependent relationship.The FTY-720 group and SCH772984 group were consistent with the GE group.3.5 Effects of GE on the expression and activation of key protein molecules in S1 P signaling pathway induced by TNF-? in HUVECP-Erk1/2,P-SphK1 and SphK1 proteins were significantly expressed in the TNF-? group,compared with the control group(P<0.01).After treatment with different concentrations of GE,the protein expression levels of P-Erk1/2,P-SphK1 and SphK1 were decreased,compared with TNF-? group(P<0.05),and consistent with FTY-720 group and SCH772984 group.3.6 The effect of HUVEC conditioned medium on proliferation of RASFsThe proliferation level of RASFs in TNF-? conditioned medium group was significantly higher than that in control conditioned medium group(P<0.01).The proliferation level of RASFs in different concentrations of GE conditioned medium group decreased to different degrees.The 50 and 100 ?g/mL GE conditioned medium group was significantly lower(P<0.01).FTY-720 conditioned medium group and SCH772984 conditioned medium group also significantly inhibited the proliferation of RASFs(P<0.01),which showed statistical significane.3.7 Effects of HUVEC conditioned medium on the secretion of IL-1?,IL-6,IL-10 and TGF-?1 in RASFsCompared with the control conditioned medium group,the levels of inflammatory cytokines IL-1? and IL-6 in RASFs of TNF-? conditioned medium group were significantly increased,and the levels of anti-inflammatory cytokines TGF-?1 and IL-10 were significantly increased(P<0.01).the levels of inflammatory cytokines IL-1? and IL-6 in RASFs in GE conditioned medium group were significantly lower(P<0.05),the levels of anti-inflammatory cytokines IL-10 and TGF-?1 were significantly increased(P<0.05),compared with TNF-? conditioned medium group.FTY-720 conditioned medium group and SCH772984 conditioned medium group were consistent with GE conditioned medium group.3.8 The Effect of HUVEC conditioned medium on key genes of Ras-Erk1/2 signaling pathway in RASFsThe expression for S1PR1 mRNA,RasmRNA and Erk1/2mRNA of RASFs in TNF-? conditioned medium group was significantly higher than that in control conditioned medium group(P<0.01).GE conditioned medium significantly decreased S1PR1 mRNA,RasmRNA and Erk1/2mRNA(P<0.01),which were consistent with the action of FTY-720 conditioned medium group and SCH772984 conditioned medium group.3.9 Effects of HUVEC conditioned medium on key protein of Ras-Erk1/2 signaling pathway in RASFsCompared with the control conditioned medium group,S1PR1,Ras and P-Erk1/2 proteins were highly expressed in RASFs of TNF-? conditioned medium group(P<0.01).The protein expression levels of S1PR1,Ras and P-Erk1/2 in RASFs of GE conditioned medium group were decreased,compared with TNF-? conditioned medium group(P<0.05).FTY-720 conditioned medium group and SCH772984 conditioned medium group were consistent with GE conditioned medium group.4 CONCLUSIONS1.The inflammatory cytokine of TNF-? induced the activation of Erk1/2,further activated SphK1,which increased the production of S1 P in HUVEC,this effect was abolished by the addition of GE.2.GE inhibited the expression of P-Erk1/2 and SphK1,and reduced the production of S1 P in HUVEC,similar to the action of P-Erk1/2 inhibitor SCH772984 and SphK1 inhibitor FTY-720,suggesting that the target of GE may be P-Erk1/2 and SphK1.3.GE treated conditioned medium of vascular endothelial cells inhibited FLS proliferation,and regulated the relative balance of proinflammatory/anti-inflammatory cytokines,at least partly associated with the ability to block the activation of SphK1/S1P/S1PR1 and its downstream Ras-Erk1/2 pathway. |
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