中药豨桐丸抗RA药效物质基础及作用机制研究

As a kind of autoimmune disease, rheumatoid arthritis is still one major problem of the medical profession to be breached. Traditional Chinese medicine compound Xitong pill, which was composed of Clerodendrum trichotomum and herba Siegesbeckiae, attending wind cold dampness evil winding resistance caused by disease, such as rheumatoid arthritis. The preliminary phytochemical research was carried out on its decomposed recipes by our group. In this project, on the base of the characteristic of Xitong pill at activating the blood circulation and dredging the collaterals, a rat model will be induced first by intracutaneous injection of Freund's complete adjuvant under the guidance of Chinese arthralgia syndrome theory about RA. Then, we will try to explore its pharmacodynamic material foundation according to the guidance of bioactivity assaying. The active component and lead compounds should be the primary focus of our research. Furthermore, we well take full advantage of chromatographic technique, such as UPLC/MS, semi-MPLC and prep-HPLC, to realize the rapid separation and enrichment drug ingredients. For the pharmacodynamic component with corroborant activity and structure, we will explore their resistance mechanisms of RA by observing the effects on the expression of platelet activating factor (PAF), platelet granule membrane protein 140 (GMP-140), platelet cluster of differentiation 40 ligand (CD40L), interleukin (IL-1, IL-6, IL-17, IL-10) and tumor necrosis factor . This work will become a significant reliable research foundation for better development and utilization of this medicine resource and the new drug R&D of traditional Chinese medicines.

作为一种自身免疫性疾病，类风湿性关节炎仍然是医学界亟待攻破的难题之一。中药复方制剂豨桐丸，由臭梧桐和豨莶草组成，主治风寒湿邪阻络所致类风湿性关节炎。在中医痹症理论的指导下，结合豨桐丸活血通络的特点，以及我们对该处方的前期拆方化学成分研究工作基础，本课题确定建立经典的佐剂性关节炎大鼠模型，进行活性跟踪下的抗RA药效物质基础研究，重点关注活性组分和先导性成分，充分利用UPLC/MS、semi-MPLC和prep-HPLC等技术，实现药效成分的快速分离富集；对于活性和结构确证的药效物质，进一步观测其对模型大鼠体内血小板活化因子(PAF)、颗粒膜糖蛋白(GMP-140)、分化抗原40配体(CD40L)、白细胞介素(IL-1、IL-6、IL-17、IL-10)和肿瘤坏死因子表达的影响，探讨其抗RA作用机制，为更好的开发和利用这一中药资源及中药新药研发提供可靠的研究基础。

中药复方制剂豨桐丸由臭梧桐和豨莶草组成，临床上用于治疗风寒湿邪阻络所致风湿性、类风湿性关节炎等症，是治疗风湿痹病的著名方剂。本项目在前期研究工作的基础上，明确了豨桐丸抗RA主要药效物质，并对其作用机制进行了探讨和研究。项目研究成果对于阐明该复方制剂抗RA药理作用机制具有重要意义，为更好的开发和利用这一中药资源及中药新药研发提供可靠的理论依据。本项目完成情况总结如下：1. 本项目采用水煎煮和乙醇热回流提取两种方式完成豨桐丸药效物质的提取，建立经典的佐剂性关节炎大鼠模型，通过检测关节炎指数、体重变化曲线、关节肿胀值及抑制率，胸腺和脾指数等指标，跟踪筛选活性部位及成分分离，确定了脂溶性提取物药效强于水提物，明确了其中所含倍半萜和二萜类活性成分为其主要的药效物质基础。2. 鉴于RA患者体内炎症因子的表达与氧化应激现象密切相关，本项目对筛选出的萜类成分进行了体外氧化应激抑制实验，结果发现倍半萜类成分表现出较强的NO抑制活性，含量较高的对映-海松烷型二萜未显示出良好的活性，而含量较低的对映-贝壳杉烷型二萜对超氧自由基和弹性蛋白酶的抑制活性显著，且部分化合物具有良好的选择性抑制活性，多数活性成分未表现出明显的细胞毒性。由此推测中药豨桐丸抗RA可能的作用机制为：多个萜类协同参与抑制了患者体内氧化应激反应，进而抑制炎症因子释放，减轻类风湿性关节炎症状。3. 构效关系研究显示：结构独特的calabrane型倍半萜4-位羰基的存在可显著增强其NO抑制活性；而豨莶草中特色的高含氧吉马烷型倍半萜内酯的9-位醚键是该类化合物重要的药效基团；对映-贝克杉烷型二萜17-位和18-位羟基的酯化对于氧化应激反应的抑制活性影响显著。在该项目资助下，共发表学术论文7篇（SCI收录3篇），会议论文2篇，获得甘肃省自然科学奖二等奖1项（第四完成人），培养博士研究生1名。