Wang Yan, Wu Hong, Gui Bin-Jie, Liu Jian, Rong Gen-Xiang, Deng Ran, Bu Yan-Hong, Zhang Heng
College of Pharmacy, Anhui University of Chinese Medicine, Qian Jiang Road 1, Hefei 230012, China; Key Laboratory of Xin'an Medicine, Ministry of Education, Hefei, 230012, China; Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Hefei, 230012, China.
College of Pharmacy, Anhui University of Chinese Medicine, Qian Jiang Road 1, Hefei 230012, China; Key Laboratory of Xin'an Medicine, Ministry of Education, Hefei, 230012, China; Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Hefei, 230012, China.
Phytomedicine. 2022 Jun;100:154068. doi: 10.1016/j.phymed.2022.154068. Epub 2022 Mar 22.
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 anti-inflammatory, anti-angiogenic and other pharmacological effects. However, the underlying mechanism through which GE affects synovial angiogenesis in RA remains unclear.
In this research, we aimed to elucidate the effect and potential mechanisms of GE on angiogenesis in RA.
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.
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.
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/ERK1/2-mediated SphK1 translocation and inhibited S1P/S1PR1 signaling activation, thereby alleviating VEGF-stimulated angiogenesis. The above evidences indicated that angiogenesis inhibition may provide a new direction for RA treatment.
类风湿关节炎（RA）是一种由促血管生成因子和抗血管生成因子失衡引起的依赖血管生成的疾病。应研究更有效的策略来阻断RA中的滑膜血管生成。栀子苷（GE）是从栀子（GJ）果实中分离出的一种天然产物，据报道具有抗炎、抗血管生成等药理作用。然而，GE影响RA滑膜血管生成的潜在机制仍不清楚。
在本研究中，我们旨在阐明GE对RA血管生成的作用及潜在机制。
通过苏木精-伊红（HE）染色、免疫组织化学（IHC）和蛋白质印迹法检测RA患者和佐剂性关节炎（AA）大鼠模型中的滑膜血管生成。通过CCK-8、EdU、Transwell、管腔形成、免疫共沉淀分析和激光扫描共聚焦显微镜检查血管内皮细胞（VECs）的生物学功能和鞘氨醇激酶1（SphK1）易位。通过在细胞和小鼠中转染SphK1-siRNA评估SphK1基因对血管生成的影响。通过在AA小鼠模型中进行基质胶栓实验测量GE对VEGF诱导的血管生成的影响。
GE有效抑制滑膜血管生成并缓解疾病进程。SphK1作为一种新的调节分子，在调节VEGF/VEGFR2和S1P/S1PR1信号方面具有潜在的重要关系。SphK1易位通过VEGFR2/PKC/ERK1/2途径被激活，并与VECs的生物学功能密切相关。GE显著降低SphK1易位，从而改善VECs的异常生物学功能。此外，在VECs和C57BL/6小鼠中转染SphK1 siRNA后，沉默SphK1有效减弱了VEGF诱导的VEC生物学功能和血管生成。在体内，基质胶栓实验表明GE显著抑制周细胞覆盖、基底膜形成、血管通透性和纤维蛋白原沉积。
我们的研究结果表明，GE通过减少SphK1易位抑制VEGF诱导的VEC生物学功能和血管生成。总体而言，研究表明GE下调VEGFR2/PKC/ERK1/2介导的SphK1易位并抑制S1P/S1PR1信号激活，从而减轻VEGF刺激的血管生成。上述证据表明血管生成抑制可能为RA治疗提供新的方向。
