To Explore The Mechanism Of Geniposide Inhibiting RA Angiogenesis From VEGF-induced Excessive SphK1 Translocation To Cell Membrane

BACKGROUND:Rheumatoid arthritis（RA）is a complex disease with multiple clinical features.Angiogenesis runs through the whole process,and the abnormal activation of vascular endothelial cells（VECs）is considered to be the key link in the occurrence and development of RA.In the study of the pathogenesis of angiogenesis,vascular endothelial growth factor（VEGF）and sphingosine 1-phosphate（S1P）signaling pathway have attracted more and more attention.However,previous studies have paid more attention to the angiogenesis promoting effects of VEGF/VEGFR2 and S1P/S1PR1 signaling pathways alone,without considering the potential relationship between them.At present,There is little known about the relationship between RA angiogenesis signaling pathways,which needs to be further explored.Traditional Chinese medicine（TCM）believes that RA belongs to"Bi syndrome"and"collaterals disease",which is the representative disease syndrome of"new disease entering the collateral".The basic theory of TCM that syndrome differentiation from collaterals should be used to treat RA.In addition,the classification of collaterals which connect channels in TCM has certain similarities with the microvascular branches in modern medicine,and the collaterals theory and angiogenesis theory are highly unified.RA collateral disease is closely related to angiogenesis in modern medicine.The Gardenia jasminoides Ellis has the effects of purging fire and removing annoyance,clearing heat and expelling damp,and cooling blood and detoxifying.Geniposide（GE）is the main active ingredient extracted and purified from the dried and mature fruit of Gardenia jasminoides Ellis,which has many pharmacological effects,including anti-RA,anti-inflammatory and immune regulation.The research group has previously confirmed that oral administration of GE has obvious anti-RA inflammatory effects.But does GE have anti-angiogenic effects?What is the mechanism of its anti-RA angiogenesis effect?All these questions need further exploration.On the basis of previous work,this project established a number of experimental models in vitro and in vivo to study the relationship between GE,angiogenesis and RA.At the same time,the anti-RA target of GE was verified by microscale thermophoresis（MST）and molecular docking experiments,and the relationship between the target and RA was explored and its role in VEGF/VEGFR2 and S1P/S1PR1 signaling pathways was clarified.We hope to provide innovative exploration for GE anti-RA.OBJECTVE:1.To explore the relationship between the abnormal biological function of VECs and the signal pathways of VEGF/VEGFR2 and S1P/S1PR1,and to clarify that VEGF induces Sph K1 translocation through VEGFR2/PKC/ERK1/2 pathway,activates S1P/S1PR1 pathway,mediates the change of biological function of VECs and triggers angiogenesis in RA.2.To explore the targeted combination of GE with Sph K1,reveal the regulation of GE on Sph K1 kinase activity and translocation behavior,clarify the new mechanism of GE targeted inhibition of Sph K1,blocking the connection between VEGF/VEGFR2 and S1P/S1PR1 signal pathways,and inhibiting RA angiogenesis.METHODS:1.The AA rat model was established and randomly divided into AA group,GE group and MTX group,while the normal group was used as a control.The joint swelling degree,arthritis index,exercise ability and rheumatoid factor（RF）were evaluated;HE staining was used to observe synovial histopathology;ELISA was used to detect the levels of VEGF,Ang-2,AS and ES in rat serum;Immunohistochemistry and Western blotting were used to detect Vimentin,CD31,VEGF,p-VEGFR2,Sph K1,p-Sph K1 and S1PR1 expression levels;The correlation analysis was used to analyze the relationship between VEGF,p-VEGFR2,Sph K1,p-Sph K1 S1P,S1PR1 and CD31expression levels.2.The VEGF-induced HUVECs angiogenesis model was established,and the expression of p-VEGFR2 and intracellular and intracellular S1P were used as the model evaluation index;CCK-8,Edu and LDH assays were used to detect HUVECs activity and drug toxicity;The cell scratch and transwell assays were used to detect HUVECs horizontal migration,vertical migration and permeability ability;The cell adhesion kit was used to detect HUVECs adhesion ability;The cell tube formation assay was used to detect HUVECs tube formation ability;The rat aortic ring experiment was used to detect VECs sprouting ability;The ELISA was used to detect HUVECs secretion of S1P;The VEGF-induced VEGFR2/PKC/ERK1/2/Sph K signaling pathway-related protein and m RNA expression and Sph K1 cytoplasmic membrane distribution were detected by WB and RT-q PCR,respectively;The co-expression of p-ERK1/2 and Sph K1 was detected by laser confocal and immunoprecipitation.3.The Sph K1 silenced HUVECs and C57BL/6 mice models were established,and the protein and m RNA expression levels of Sph K1 in HUVECs and synovial tissue were used as model evaluation indicators;CCK-8,Edu and LDH experiments were used to detect the effect of Sph K1 silencing on VEGF-induced HUVECs activity and drug toxicity;The cell scratch and transwell assays were used to detect the effect of Sph K1 silencing on VEGF-induced HUVECs horizontal migration,vertical migration and permeability;The cell adhesion kit was used to detect the effect of Sph K1 silencing on VEGF-induced HUVECs adhesion ability;The cell tube formation assay was used to detect the effect of Sph K1 silencing on VEGF-induced HUVECs tube formation ability;The rat aortic ring experiment was used to detect the effect of Sph K1 silencing on VEGF-induced VECs sprouting ability;The ELISA was used to detect the effect of Sph K1 silencing on VEGF-induced HUVECs secretion of S1P;The HE staining was used to observe the pathology of matrigel plug;The immunohistochemistry was used to detect the expression level of CD31;The immunofluorescence was used to detect pericyte coverage,basement membrane formation,vascular permeability and fibrinogen accumulation.4.The HEK293 model transfected with the Sph K1 phosphorylation single point mutation plasmid was established,and the cells were screened with G418 sulfate solution for 7 days.At the same time,the protein and m RNA expression levels of Sph K1 in HEK293 were used as the model evaluation indicators;The WB and ELISA were used to detect Sph K1 cytoplasmic and membrane distribution and the expression of Sph K1,p-Sph K1,S1PR1 proteins and S1P cytokines.5.Study on GE targeted binding to Sph K1:The MST was used to detect the binding ability of GE and Sph K1 protein;The Autodock Vina molecular simulation was used to detect the possible binding targets of GE and Sph K1;The CESTA was used to detect the binding of GE and Sph K1 in cells,The ADP-Glo^TMkinase detection kit was used to detect the inhibition of Sph K1 kinase activity by GE.RESULTS:1.The VEGF/VEGFR2 and S1P/S1PR1 signalings mediated RA angiogenesis were verified in vivo and GE interventionThe neovascularization was found in the synovium of AA rats and RA patients,accompanied by abnormal activation of VEGF/VEGFR2 and S1P/S1PR1 signal pathways.The GE（60 mg/kg/day）and MTX（0.5 mg/kg/3days）can effectively reduce the degree of joint swelling,arthritis index and RF level of AA rats and restore the exercise ability of rats;They can restore the balance of pro-angiogenic factors VEGF,Ang-2 and anti-angiogenic factors AS,ES in rat;They can improve the pathological condition of rat synovium in varying degrees;Both can inhibit the abnormal activation of VEGF/VEGFR2 and S1P/S1PR1 signaling pathways;Further studies showed that Sph K1 was abnormally expressed in RA synovium and was closely related to synovium angiogenesis;The above results showed that there is angiogenesis in RA synovium,and GE can effectively alleviate the symptoms of AA.2.The study on the biological function changes and mechanism of VECs induced by VEGFVEGF（20 ng/m L）-induced HUVECs model was constructed in vitro with VEGFR2 inhibitor（Apatinib,5μM）,PKC inhibitor（Go 6983,5μM）,ERK1/2inhibitor（SCH772984,1μM）and Sph K1 inhibitor（PF-543,5μM）intervention for24 h,and it was found that each group of inhibitors could inhibit the changes of VEGF-induced HUVECs proliferation,migration,permeability,adhesion,tube formation,budding ability,and S1P secretion function to varying degrees.Further research found that VEGF can mediate the activation of Sph K1 through VEGFR2/PKC/ERK1/2 signaling,promote its translocation,induce the activation of S1P/S1PR1 signaling,and participate in the change of HUVECs biological function.The mechanism of VEGF-induced Sph K1 activation showed that there was a direct interaction between p-ERK1/2 and Sph K1 in HUVECs cells.Under the stimulation of VEGF,the interaction between p-ERK1/2 and Sph K1 was significantly enhanced,After Apatinib,Go 6983,SCH772984 and PF-543 intervention,the effect between p-ERK1/2 and Sph K1 was reduced to varying degrees.The above results showed that VEGF-induced Sph K1 translocation was involved in the changes of VECs biological function.3.The effect of Sph K1 gene silencing on VEGF-induced angiogenesis in vivo and in vitro and exploration of Sph K1 phosphorylation site activityThe HUVECs was transfected with Sph K1-si RNA（20 n M）to observe the effect of Sph K1 silencing on the biological function of VEGF-induced VECs in vitro.The results showed that Sph K1 silencing could effectively alleviate the changes of HUVECs proliferation,migration,permeability,adhesion,tube formation and S1P secretion induced by VEGF;Sph K1-si RNA（5μg/animal）was injected into C57BL/6mice to observe the effect of Sph K1 silencing on VEGF induced angiogenesis in vivo.The results showed that Sph K1 silencing could significantly change the structure（pericyte coverage and basement membrane formation）and function（permeability and fibrinogen accumulation）of VEGF induced angiogenesis;The Sph K1phosphorylation single point mutant plasmid（5μg/well）was transfected into HEK293 to observe the effect of different phosphorylation sites of Sph K1 on the mechanism of VEGF-induced Sph K1 activation in vitro.The results showed that Sph K1-Ser225 phosphorylation site could affect the translocation of Sph K1 induced by VEGF and the activation of S1P/S1PR1 signal.Sph K1-Thr193 phosphorylation site may affect the phosphorylation of Sph K1-Ser225 induced by VEGF,affects the distribution of cell membrane and the activation of S1P/S1PR1 signal.The above results showed that Sph K1-Ser225 site has an important effect on Sph K1translocation and S1P/S1PR1 signal activation.4.GE targeted inhibition of VEGF-induced Sph K1 translocation to alleviate RA angiogenesisThe intervention results of GE（25,50,100μmol/L）on VEGF-induced HUVECs showed that GE could alleviate the proliferation,migration,permeability,adhesion,tube formation,budding and S1P secretion ability of VEGF-induced HUVECs by regulating the excessive Sph K1 translocation;The GE（120 mg/kg）was used to intervene VEGF-induced angiogenesis in AA mice.The results showed that GE could significantly change the structure（pericyte coverage and basement membrane formation）and function（permeability and fibrinogen accumulation）of VEGF induced angiogenesis in matrigel plug;In the study of GE targeted binding to Sph K1,the MST results showed that the dissociation constant Kd of GE and Sph K1 was34.26±0.97μM,which indicated that GE was directly bound to Sph K1 protein.The Auto Dock Vina molecular docking simulation results showed that the binding energy of GE and Sph K1 is-8.3 kcal/mol,the binding site is the kinase catalytic domain of Sph K1,and the binding sites are Asn-22,Ser-79,Asp-81,Gly-82,Glu-86,Ala-110and Gly-343,respectively.The CESTA experiment results showed that GE could bind to endogenous Sph K1 protein and increase its thermal stability,but GE could not bind to Sph K1 protein with A22G-S79A-A81G-G82A-G86A-A110G-G343A site mutation.The ADP-Glo^TMkinase assay results showed that the IC₅₀of GE against 125 ng of Sph K1 protein was 3.856μM in the reaction system of 25μM ATP+ADP.All the above results confirmed that GE could alleviate RA angiogenesis by targeting VEGF-induced Sph K1 translocation.CONCLUSION:1.RA is an angiogenesis-dependent disease,and there is abnormal activation of VEGF/VEGFR2 and S1P/S1PR1 pro-angiogenesis signaling pathways in synovial tissue.2.VEGF induces Sph K1 translocation through VEGFR2/PKC/ERK1/2 signal pathway,and then activates S1P/S1PR1 pathway to participate in the changes of VECs biological function;The treatment with GE can down-regulate the expression of Sph K1,weaken the Sph K1 translocation and inhibit the activation of S1P/S1PR1signal pathway,and alleviate the changes of biological function of VECs induced by VEGF.3.VEGF-induced phosphorylation of Sph K1 at Ser225 has important effects on Sph K1 translocation and activation of S1P/S1PR1 signaling.4.GE can directly inhibit the kinase activity of Sph K1 and downregulate the translocation of Sph K1.The key active sites for the interaction between GE and Sph K1 may exist in Asn-22,Ser-79,Asp-81,Gly-82,Glu-86,Ala-110 and Gly-343.The molecular mechanism of GE inhibiting Sph K1 translocation may be involved in alleviating the VEGF-induced VECs abnormal biological function.