土家药白三七抗类风湿性关节炎血管新生作用物质基础及TLR4-NF-κB通路介导的作用机制研究

As an autoimmune disease, rheumatoid arthritis (RA) has caused great pain to the patients, however, it is still a challenge problem in the world to treat this disease. Tujia ethnomedicine Baisanqi (Panax japonicus C. A. Mey) was recorded to possess the function of expelling wind-evil and removing wetness-evil, regulating “qi” and alleviating pain, make it commonly used in the treatment of wind-dampness, arthralgia-syndrome and various arthritis diseases. However, the therapeutic material basis and mechanism of its anti-RA effect are not clear. In our previous studies, saponins including three polyacetylenic oleanane-type triterpenoids were isolated from Baisanqi, and some of the saponins can inhibit the proliferation and migration of Ang2 induced VSMCs. So the hypothesis was put forward that saponins are the material basis on anti-RA of Baisanqi, and its mechanism may be related to reducing the inflammation of synovium and inhibiting angiogenesis through TLR4-NF-κB pathway. In this study, we aimed to separate the novel saponins guided by the activity of anti proliferation for RA-FLS; the combined screening model of in vitro and in vivo will implement to study the effects of saponins from Baisanqi in alterations of genes, proteins and cytokines which played key roles in pro-angiogenesis and pro-inflammatory in RA, which mediated by TLR4-NF-κB pathway; to clarify the material basis of efficacy and molecular mechanism for Baisanqi in the treatment of RA. This project will provide scientific foundation for the application of this ethnomedicine in treatment of wind-dampness, arthralgia-syndrome, and various arthritis diseases.

类风湿性关节炎（RA）是一种自身免疫性疾病，给患者造成了极大的痛苦，但其治疗仍是世界医学界亟待攻破的难题。湘西北土家药白三七具扶正祛邪、祛风除湿、行气止痛之功效，常用于治疗风湿痹症、关节炎等，但其抗RA的药效物质基础和作用机理尚不明确。课题组从白三七中分离到皂苷类成分，尤其是带有罕见人参炔醇基的皂苷类成分，部分成分可抑制血管紧张素II诱导的大鼠血管平滑肌细胞增殖与迁移。前期工作提示白三七皂苷可能通过TLR4-NF-κB通路抑制RA血管新生，减轻关节滑膜炎症而发挥抗RA作用。本课题拟用体外抑制RA成纤维样细胞体内增殖活性为导向，运用现代分离手段分离其中结构新颖的皂苷类成分，利用体外筛选相结合的模式，对白三七总皂苷在TLR4-NF-κB通路中对促炎和促血管生成中起关键作用的基因、蛋白和细胞因子的变化进行研究，阐明白三七RA的药效物质基础和作用机制，为该民族药治疗风湿痹症的应用奠定基础。

土家药白三七为五加科（Araliaceae）人参属（Panax）植物竹节参（Panax japonicus C. A. Mey）的干燥根茎，又名竹节人参、竹节三七等。白三七素有“土家族药神参”之称，属珍稀濒危的名贵“七类”中草药，其性温，味甘、微苦，具有滋补强壮、散瘀止痛、止血、祛痰等功效。.本研究综合运用现代色谱分离方法对白三七化学成分进行了系统的研究，从中分离并鉴定了52个化合物（45个皂苷类成分），其中6个为新化合物，包括3个带有人参炔三醇基的人参皂苷类成分，研究结果进一步丰富了白三七次生代谢产物的多样性，为新的生物活性的发现奠定了物质基础。对白三七醇提物进行了抗RA活性及机制研究，但暂未发现其显著抗RA活性。运用MTT法对所分离单体化合物的细胞毒活性和抗类风湿性关节炎活性进行了筛选，发现部分化合物具中等强度的抗肿瘤活性，为其进一步临床应用提供了科学研究基础。.开发了负载土家药白三七中单体化合物竹节参皂苷IVa 甲酯的仿生纳米药物传递系统 (LSCMR NPs)。该系统安全低毒，具有较好的生物相容性，能延长载药系统的血液循环时间，逃避免疫监视，靶向富集于肿瘤部位，CSME的细胞杀伤能力和Ce6的光动力协同作用能有效抑制乳腺肿瘤的生长。LSCMR靶向肿瘤的纳米给药平台为提高天然药物的抗肿瘤效率提供了研究基础。.建立了能对甲酰胺嘧啶DNA糖基化酶进行灵敏检测的荧光生物传感器，该生物传感器能在大肠杆菌中对甲酰胺嘧啶DNA糖基化酶进行成像，利用此成像发掘了土家药中分离的化合物人参皂苷Re对大肠杆菌甲酰胺嘧啶DNA糖基化酶的激活作用，人参皂苷Re能清除大肠杆菌中H2O2诱导产生的ROS，说明其具有氧化损伤修复作用。研究为天然来源化合物活性多样性的筛选提供了新方法。