栀子苷双靶调控SphK1-S1P-S1PR1和Ras-Erk1/2信号转导干预类风湿性关节炎滑膜微环境炎症反应

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia, angiogenesis, formation of pannus and erosion of articular cartilage and bone. It belongs to the category of “arthralgia” in traditional medicine. The imbalance of synovial microenvironment plays an important role in the development and progression of RA. Fibroblast synovial cells (FLS) and vascular endothelial cells (VEC) are the main participants in synovial microenvironment. The functional changes of FLS and VEC are closely related to the entire course of RA. Sphingosine-1-phosphate (S1P) is a product of activation of sphingosine kinases (SphKs), which is a kind of lysophospholipid with a wide range of biological activities. After its binding with its receptor S1PRs which belong to G protein-coupled receptors (GPCRs), it participates in cell proliferation, apoptosis and infiltration of inflammatory factors through a variety of pathways and plays an important role in autoimmune diseases. But so far, the signaling pathways that regulate the inflammatory mechanisms of RA are not well understood. Our previous studies found that geniposide (GE), an iridoid glycoside obtained from Gardenia jasminoides Ellis, had significant anti-inflammatory effects and inhibitory effects on FLS abnormal proliferation in rats with adjuvant arthritis (AA), and down-regulated the abnormal expression of p-Erk1/2, p-SphK1 and S1PR1 proteins in FLS of AA rats. Based on a good previous experimental foundation and a large number of literature research, we propose the following scientific hypothesis: “GE dual-target regulates the over-phosphorylation of SphK1 and Erk1/2, regulates SphK1-S1P-S1PR1 and downstream GPCRs mediated Ras-MEK1/2-Erk1/2 signal transduction, inhibits abnormal proliferation and angiogenesis of FLS, interfers FLS and VEC inflammatory response and VEC biological function changes, which plays an important role in the treatment of RA”. This topic intends to use the activation and inhibition of signal transduction, gene overexpression and gene silencing, construction and transfection of mutant plasmids, co-immunoprecipitation, immunofluorescence, flow cytometry, ultra performance liquid chromatography-mass spectrometry, RT-qPCR, Western blotting and cell co-culture, and other key technologies to explore the inflammatory pathological mechanism of RA in a deeper level, to reveal the specific target of GE double target regulation and to explain the scientific connotation of Traditional Chinese Medicine from "collaterals" to treat arthralgia, and to lay the foundation for the construction of a new drug target for the treatment of RA.

类风湿性关节炎(RA)是以滑膜炎性增生、血管新生、血管翳形成并侵蚀关节软骨和骨为病理特征的自身免疫性疾病。滑膜微环境主要参与者成纤维滑膜细胞(FLS)和血管内皮细胞(VEC)功能改变与其密切相关；鞘氨醇激酶(SphKs)激活的1-磷酸鞘氨醇(S1P)结合受体S1PR参与细胞增殖、炎症反应，在RA病程中发挥重要作用。但调控炎症机制的信号通路尚不明确。课题组研究发现栀子苷(GE)下调FLS中p-Erk、p-SphK1和S1PR1异常高表达。提出假说：GE双靶调控SphK1和Erk过度磷酸化，调节SphK1-S1P-S1PR1及Ras-Erk信号转导，抑制FLS异常增殖和血管新生，干预炎症反应及VEC功能改变。拟采用基因过表达和沉默、突变质粒构建和转染、细胞共培养等关键技术，探究RA炎症机制，揭示GE双靶调控具体靶点，阐释中医药从“络”治痹理论的科学内涵，为构建治疗RA新的药物作用靶点奠定基础。

类风湿性关节炎(RA)是一种以滑膜组织慢性炎性增生、血管新生、血管翳形成为病理特征的难治性自身免疫性疾病，成纤维滑膜细胞(FLSs)的异常增殖和血管内皮细胞(VECs)的血管新生协同促进特征性血管翳形成。前期研究发现清热解毒中药栀子有效成分栀子苷(GE) 对佐剂性关节炎(AA)大鼠有显著的抗炎作用，抑制FLSs异常增殖，下调p-Erk1/2过度表达，调控Ras-MEK1/2-Erk1/2信号通路激活。建立TNF-α诱导MH7A及HUVECs炎症损伤模型，分别制备来自于MH7A及HUVECs的符合细胞体外培养基本要求的条件培养基，探讨FLSs与VECs在RA关节滑膜炎性微环境中相互作用的物质基础和作用机制以及GE的干预作用，研究证实MH7A可能是通过活化S1P-S1PR1 和下游 RhoA-F-actin-NF-κB信号通路而改变HUVECs生物学功能，GE抑制MH7A对HUVECs的诱导作用，可能是通过抑制S1P-S1PR1和RhoA-NF-κB-F-actin信号通路的激活，发挥抑制血管新生作用；而HUVECs可能通过增加 S1P分泌量，介导MH7A中S1PR1对Ras-Erk1/2信号通路的调控而促进MH7A异常增殖，GE显著抑制MH7A异常增殖，降低S1PR1、Ras和P-Erk1/2的蛋白表达水平，阻断S1PR1对Ras-Erk1/2信号通路的调控，恢复促/抑炎细胞因子间动态平衡，抑制炎症发展。进一步发现GE下调HUVECs中S1P分泌水平，可能与其抑制P-Erk1/2和SphK1共表达相关，且与P-Erk1/2和SphK1抑制剂具有相似的作用，提示GE双靶抑制SphK1和Erk1/2过度磷酸化，阐释其通过抑制滑膜异常增生和血管新生，治疗RA关节滑膜微环境炎症反应的作用机制。为构建筛选治疗RA新的药物作用靶点提供技术平台，也为清热解毒中药研究与应用提供更多实验依据。