Geniposide alleviates VEGF-induced angiogenesis by inhibiting VEGFR2/PKC/ERK1/2-mediated SphK1 translocation

Background: Rheumatoid arthritis (RA) is an angiogenesis-dependent disease caused by the imbalance of pro-and anti-angiogenic factors. More effective strategies to block synovial angiogenesis in RA should be studied. Geniposide (GE), a natural product isolated from the fruit of Gardenia jasminoides Ellis (GJ), is reported to have antiinflammatory, anti-angiogenic and other pharmacological effects. However, the underlying mechanism through which GE affects synovial angiogenesis in RA remains unclear. Purpose: In this research, we aimed to elucidate the effect and potential mechanisms of GE on angiogenesis in RA. Materials and methods: Synovial angiogenesis in patients with RA and a rat model of adjuvant arthritis (AA) was detected by hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), and western blottiing. The biological functions of vascular endothelial cells (VECs) and sphingosine kinase 1 (SphK1) translocation were checked by CCK-8, EdU, Transwell, tube formation, co-immunoprecipitation assays, and laser scanning confocal microscopy. The effect of the SphK1 gene on angiogenesis was assessed by transfection of SphK1-siRNA in cells and mices. The effect of GE on VEGF-induced angiogenesis was measured by Matrigel plug assay in a mouse model of AA. Results: GE effectively inhibited synovial angiogenesis and alleviated the disease process. SphK1, as a new regulatory molecule, has a potentially important relationship in regulating VEGF/VEGFR2 and S1P/S1PR1 signals. SphK1 translocation was activated via the VEGFR2/PKC/ERK1/2 pathway and was closely linked to the biological function of VECs. GE significantly reduced SphK1 translocation, thereby ameliorating the abnormal biological function of VECs. Furthermore, after transfection of SphK1 siRNA in VECs and C57BL/6 mice, silencing SphK1 caused effectively attenuation of VEGF-induced VEC biological functions and angiogenesis. In vivo , the Matrigel plug experiment indicated that GE significantly inhibited pericyte coverage, basement membrane formation, vascular permeability, and fibrinogen deposition. Conclusions: Our findings suggest that GE inhibited VEGF-induced VEC biological functions and angiogenesis by reducing SphK1 translocation. Generally, studies have revealed that GE down-regulated VEGFR2/PKC/ERK1/2mediated SphK1 translocation and inhibited S1P/S1PR1 signaling activation, thereby alleviating VEGFstimulated angiogenesis. The above evidences indicated that angiogenesis inhibition may provide a new direction for RA treatment.

Automated summary

The study found that geniposide can inhibit angiogenesis in rheumatoid arthritis and alleviate disease progression. The research also revealed the important role of SphK1, VEGF/VEGFR2, and S1P/S1PR1 signaling pathways in the effect of geniposide on synovial angiogenesis.