冷蒿中新颖二聚倍半萜类成分及其抗类风湿关节炎作用研究

Development of novel effective immunosuppressants demands urgently, since rheumatoid arthritis is harm to human health severely. It is an important strategy to explore effective lead compounds with low toxicity from natural medicinal plants. Triditionally, it has the effects of detumescence and pain relief. In our preliminary study, some dimeric sesquiterpenoids were isolated from Artemisia frigida and two of them exhibited significant inhibition effects on NO production and NF-κB signaling pathway with their IC50 values of 1.65 and 3.87 μM respectively. On the basis of the previous research, we continue to aim at discovering structurally novel dimeric sesquiterpenoids derived from A. frigida and find bioactive target compounds supplemented by activity evaluation using the model of macrophages in vitro. Meanwhile, the therateutic effects of target compounds were evaluated on collagen-induced arthritis (CIA) rat model. The in vitro anti-inflammatory effects of them will be investigated by assaying their influence on inflammatory cytokines and the expression of related target genes in NF-κB signaling pathway with molecular biology techniques like PCR and western-blot. The implementation of this research will further explore the resources and expand the scope of clinical application of A. frigida, and meanwhile it can provide beneficial exploration to discover novel drugs for the treatment of RA.

类风湿关节炎（RA）严重危害着人类健康，开发新型高效的免疫抑制剂迫在眉睫。从天然药用植物中筛选高效低毒的先导化合物，是进行抗RA药物研究的重要策略之一。冷蒿具有消肿、止痛之功效。前期研究中，我们从冷蒿提取物的氯仿萃取部位分离鉴定了多个二聚倍半萜，体外实验表明其中两个新化合物具有明显抑制NO释放（IC50分别为1.65和3.87 μM）和NF-κB通路的活性。本项目拟在此基础上，进一步对冷蒿中的二聚倍半萜化合物进行系统定向分离鉴定。通过体外巨噬细胞为模型进行活性评价，发现活性显著的目标化合物。同时以CIA大鼠作为体内药效筛选模型，采用qRT-PCR、Westernblot、细胞转染等分子生物学技术，观察目标化合物对NF-κB通路中各个炎症细胞因子及其相关靶基因表达的影响。本研究的顺利实施将拓展冷蒿的临床应用范围，并进一步挖掘冷蒿资源，同时为发现新型抗类风湿关节炎药物提供有益探索。

冷蒿作为传统的中药和民族药，其味苦，性凉。具有止血、消肿、止痛之功效，常用于治疗类风湿关节炎。本研究结合体内动物实验，从冷蒿种分离鉴定出34个化合物，其中含有7个新化合物。其类型主要包括黄酮、二萜、倍半萜、木脂素和三萜等。其中新化合物和新骨架化合物的发现，更加深入地挖掘和丰富了冷蒿次生代谢产物的多样性。对部分化合物进行了抗炎活性筛选，其中化合物3，4在20μM浓度下，表现出较显著TNF-α和IL-6抑制活性。另外，单体化合物24对油酸诱导的HepG2细胞的脂质积累具有有效的抑制作用，且无细胞毒性。结果表明，其能降低与脂肪生成相关的SREBP1、ACC和FASN基因的表达水平，增加了与脂肪酸氧化相关的3个基因(PPARα、ACOX1和FGF21)的表达水平。此外，SREBP1、PPARα和FGF21蛋白水平也观察到了类似的结果。PPARα拮抗剂GW6471可部分逆转(+)-dehydrovomifoliol的作用。基于其通过PPARα-FGF21轴对肝脏脂肪生成和脂肪酸氧化信号通路的影响，为阐明冷蒿在传统临床疗效包括抗类风湿关节炎活性的物质基础和作用机制方面，提供有益探索。同时，冷蒿的小鼠体内动物实验HE结果显示其具有一定的心脏毒性。本研究有助于冷蒿药效物质基础的阐明并为基于冷蒿来源的药物分子研发提供坚实的科学依据。