Exploring The Role And Mechanism Of Geniposide In Regulating Experimental Arthritic Angiogenesis From S1P-induced Ang-2 Exocytosis

Rheumatoid arthritis（RA）is an inflammatory disease with angiogenesis as its main pathological feature.Angiogenesis promotes synovial inflammation and pannus formation,aggravates the destruction of articular cartilage and bone,and plays a key role in RA progression.Angiopoietin-2（Ang-2）,a marker of endothelial cell activation,can induce endothelial destabilization,triggering angiogenesis.Sphingosine 1-phosphate（S1P）is a phospholipid signaling molecule that is catalyzed by sphingosine kinases（Sph Ks）and is transferred extracellularly via a transporter on the plasma membrane to bind to its receptor S1PR1/3,which activates the coupled Gαi and Gαq,causing activation of phospholipase Cβ3（PLCβ3）.Phosphatidylinositol 4,5-bisphosphate（PIP₂）on the cell membrane is hydrolysed by PLCβ3 to inositol 1,4,5-trisphosphate（IP₃）and diacylglycerol.IP₃ diffuses into the cytoplasm and binds to the Ca²⁺receptor channel inositol 1,4,5-trisphosphate receptor（IP₃R）on the endoplasmic reticulum to induce Ca²⁺release,which triggers Ang-2 exocytosis in the Weibel-Palade body（WPB）.However,the way in which WPB exocytosis of Ang-2 is unknown.Exocytosis is a prerequisite for Ang-2 to play its role,so it is of great significance to investigate the mechanism and mode of Ang-2 exocytosis.Geniposide（GE）is the main effective component of Gardenia jasminoides Ellis,which plays antiangiogenesis and anti-inflammatory effects.Previous studies have revealed that GE could lower the serum level of Ang-2 in adjuvant arthritis（AA）rats and inhibit the activation of S1P/S1PR1/3 signal.However,whether and how GE regulates Ang-2exocytosis remains unclear.OBJECTIVETo explore the effect of S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis on Ang-2 exocytosis and biological function of human umbilical vein endothelial cell（HUVEC）.To elucidate the new mechanism by which GE negatively regulates S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis,intervenes Ang-2 exocytosis,improves the biological function of HUVEC,and inhibits RA angiogenesis.METHODSThe AA rat model was established.GE and MTX were used to intervene.Arthritis index,global assessment,paw swelling and swollen joint count were used to evaluate the model.HE staining was used to observe the synovial pathologic morphology of AA rats.Color Doppler ultrasound was applied to observe signal of synovial blood flow in knee joint of AA rats.Immunohistochemistry was used to determine CD31,vascular endothelial growth factor（VEGF）,S1PR1,S1PR3,PLCβ3,IP₃R and Ang-2 levels in the synovial tissues of AA rats.The influence of GE on angiogenesis in adjuvant arthritis was further investigated by matrigel plug assay.S1P-induced HUVEC was used as the research object.ELISA,immunofluorescence and Western blot were used to dynamically monitor Ang-2 exocytosis,and transmission electron microscopy was used to observe Ang-2 exocytosis pattern.The HUVEC was treated with GE,S1PR1/3 inhibitor VPC23019 and IP₃R inhibitor 2-APB.ELISA and immunofluorescence were used to detect the secretion of Ang-2.ELISA was applied to detect intracellular IP₃ content.Flow cytometry was used to detect intracellular free Ca²⁺level.RT-qPCR was applied to detect the m RNA levels of S1PR1,S1PR3,PLCβ3,IP₃R and Ang-2.Western blot was used to assess protein expressions of S1PR1,S1PR3,PLCβ3and IP₃R.Furthermore,the proliferation,migration and lumen formation capacity of HUVEC were determined to evaluate the regulatory effect of GE on Ang-2 exocytosis and the biological function of HUVEC through S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis.RESULTS1.The regulatory effect of GE on angiogenesis in experimental arthritisGE（60,120 mg/kg）could significantly reduce the arthritis index,global assessment,paw swelling and swollen joint count of AA rats,improve the pathological morphology of synovium,reduce the synovium blood flow signal of knee joints and the expression of CD31 and VEGF.The levels of S1PR1,S1PR3,PLCβ3,IP₃R and Ang-2 were high in synovial tissues of AA rats.GE administration intervention could remarkably down-regulate the expression of these proteins.These results suggested that the S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis in the synovial tissues of AA rats may be activated to induce Ang-2 exocytosis and thus trigger angiogenesis.The results of matrigel plug assay showed that angiogenesis was more obvious in AA group than in the control group.GE intervention reduced the formation of new blood vessels.These results suggested that GE may play a role in the treatment of RA by regulating the S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis and inhibiting angiogenesis.2.Study on the regulation of Ang-2 exocytosis by S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis and GE interventionS1P induced rapid exocytosis of Ang-2 in HUVEC cells.When S1P（1μM）acted for 30minutes,Ang-2 was secreted in large quantities,and the WPB were fused to form vesicles,suggesting that the Ang-2 exocytosis may be multigranular exocytosis.In S1P-induced HUVEC cells,Ang-2 exocytosis,IP₃ content and free Ca²⁺level were significantly increased.The m RNA and protein levels of S1PR1,S1PR3,PLCβ3 and IP₃R were significantly increased,while the m RNA level of Ang-2 was not significantly changed.GE,VPC23019 and 2-APB could inhibit the effect of S1P,but had no effect on the m RNA level of Ang-2.The above results confirmed that the activation of S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis could induce Ang-2 exocytosis.3.Effects of S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis on biological function of HUVEC and GE interventionCompared with the control group,the proliferation,migration and lumen formation of S1P-induced HUVEC were significantly enhanced.Compared with S1P group,GE,VPC23019 and 2-APB interventions improved the biological function changes of HUVEC.These results indicated that the S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis induced Ang-2 exocytosis,promoted the biological function changes of HUVEC,and triggered angiogenesis.It was clear that GE could interfere with Ang-2 exocytosis and inhibit the biological function of HUVEC by negatively regulating S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis,so as to play a role in inhibiting angiogenesis.CONCLUSIONS1.GE inhibits angiogenesis and plays a role in the treatment of RA.2.The activation of S1P/S1PR1/3/PLCβ3/Ca²⁺signal axis induces Ang-2 to exhibit multi-particle exocytosis,promoting the biological function changes of HUVEC and triggering angiogenesis.3.GE negatively regulates the signal axis of S1P/S1PR1/3/PLCβ3/Ca²⁺,interferes with Ang-2 exocytosis,improves the biological function changes of HUVEC and inhibits angiogenesis.