黑骨藤总苷和杠柳苷对完全弗氏佐剂诱导的大鼠类风湿关节炎保护作用研究

Guangxi has rich resources of medicinal plants, a variety of natural plants are available for the treatment of Rheumatoid arthritis（RA）. Chinese medicines comprising Periploca forrestil for treating RA have been used in clinical, Periploca forrestil contain cardiac glycosides, alkyl steroid. Periplocoside is its active ingredients, its pharmacological mechanism in treating RA is still less known. Both cell-mediated and humoral immune reactions participate in the pathogenesis of RA. RA is characterized by synovial inflammation and hyperplasia. Hyperplasia of fibroblast-like synoviacytes (FLS) exhibits features tumor-like transformation. Anchorage-independent growth, resistance to apoptotic signaling and invasiveness eventually result in cartilage and bone destroyed..In this project, we observe the protective effects of total glycosides of Periploca forrestil on bone erosion in complete Freund's adjuvant-induced rheumatoid arthritis (AA) in rats. .In humoral immunity, we observe the effects of total glycosides of Periploca forrestil on the levels of anti-CCP antibody and anti-Bhsp65 antibody in AA rats. while in cellular immunity we explore the effects of total glycosides of Periploca forrestil and Periplocoside on the gene levels of Th1, Th2, Th17 cytokines and IL-12 family cytokines in Spleen Cells and Lymph Node Cells, and on the expression of Th1, Th2, Th17 and Treg transcription factors in Lymph Node Cells in vitro and in vivo..We also observe the effects of total glycosides of Periploca forrestil and Periplocoside on the inflammatory mediator levels of COX-2, PGE2, MMP-1, MMP-13 and VEGF in FLSs in vitro and in vivo, and their effects on MAPK and JAK/STAT signaling pathway in FLSs will be evaluated in vitro and in vivo..Then we will evaluate the effects of total glycosides of Periploca forrestil and Periplocoside on the proliferation, apoptosis induction of FLSs as well as their effects on NF-κB signaling pathway. With respect to their effects on the induction apoptosis of FLSs, we will measure NF-κB-regulated gene transcription and NF-κB-regulated gene products involved in cell proliferation (cyclin D1 and COX-2), antiapoptosis (Bcl-2 and FLIP), and invasion (MMP-13 and VEGF). Their effects on NF-κB signaling pathway will be validated by measuring expression of IκBαand phosphorylated IκBα, localization activity of NF-κBp65 and DNA binding activity of NF-κB..To validate the effects of total glycosides of Periploca forrestil and Periplocoside on osteogenic differentiation of FLSs, we will culture FLS in a osteogenesis-inducing medium, ALP, mineral deposition and osteogenic differentiation markers (osteopontin and RANK) expression in cells will be detremined..The evaluation through multi-target is in line with traditional Chinese medicine treatment mechanism. This research on immunology and biology mechanism of action of Periploca forrestil will provide scientific data for the treatment of RA.

广西药用植物资源丰富，以黑骨藤为主药的祛风湿民族药已应用于临床，但其药理机制研究较少。细胞和体液免疫反应介导类风湿性关节炎（RA）发病，成纤维样滑膜细胞(FLS)肿瘤样侵蚀与RA骨损伤有关。本项目观察黑骨藤总苷对完全弗氏佐剂诱导大鼠类风湿性关节炎（AA）骨损伤作用；体液免疫上观察黑骨藤总苷对AA大鼠抗CCP抗体和抗Bhsp65抗体水平的影响；细胞免疫上观察黑骨藤总苷和杠柳苷对AA大鼠脾、淋巴结Th1、Th2、Th17细胞因子和IL-23p19、IL-27p28及淋巴结Th1、Th2、Th1 7和Treg转录因子的影响。观察黑骨藤总苷和杠柳苷对FLS中 COX-2、PGE2、MMP、VEGF水平及MAPK、JAK/STAT细胞信号转导通路的影响；对FLS增殖、细胞凋亡、诱导FLS向成骨细胞分化及对NF-κB信号通路的影响。本项目从多靶点综合评价黑骨藤作用机理可为中药治疗RA提供科学数据。

黑骨藤已被用作中国民间医学用于治疗类风湿性关节炎和创伤。黑骨藤苷（Periploca forrestii saponin , PFS）和其活性化合物杠柳苷的抗关节炎活性仍未得到证实。我们研究了黑骨藤苷对完全弗氏佐剂诱导大鼠类风湿性关节炎（AIA）骨损伤作用；在体内和体外实验，研究黑骨藤总苷和杠柳苷对AIA 大鼠脾炎症因子及相关转录因子的影响；研究黑骨藤总苷和杠柳苷对滑膜成纤维样细胞(FLS) 中炎症因子及相关转录因子的影响。 研究PFS对II型胶原蛋白（CII）诱导的关节炎（CIA）小鼠的保护作用以及研究PFS和杠柳苷调节破骨细胞发生及作用机制。此外, 我们研究甲氨蝶呤联合复方风湿宁对类风湿性关节炎滑膜成纤维细胞Treg /Th17 免疫平衡的影响。研究DNFB反复涂布诱导即时反应大鼠皮炎模型。发表论文三篇，其中，SCI两篇，北大中文核心1篇。