基于“SREBP-脂质代谢-Th17细胞分化”轴研究中药地乌皂苷抗类风湿关节炎的作用及机制

Decreasing Th17 Differentiation by regulating fatty acid metabolism has been considered to be an important strategy to prevent rheumatoid arthritis (RA). Crude triterpenoid saponins from Anemone flaccida (Di Wu, AFS) showed significant anti-inflammatory and immunosuppressive activity in phase III clinical trials, which is expected to be developed into a new drug of traditional Chinese medicine with the high efficiency and low toxicity for the treatment of RA. However, the underlying biological mechanisms remain elusive. Our previous study showed that crude triterpenoid saponins from AFS and Anhuienosides C (AC), one of the main active compounds from AFS, decreased the ratio of Th17 cells in the spleen of RA mice induced by type-I collagen (CIA). In addition, AFS and AC treatment significantly decreased Th17 differentiation, improved lipid metabolism and inhibited SREBP transcriptional activity. On the basis of previous study, we will further investigate the effect of AFS and AC on SREBP-mediated fatty acid metabolism signaling pathways and the ones involved in the regulation of Th17 differentiation by immunohistochemistry, luciferase assay, western blot and siRNA analysis in Scapfl/fl knockout-mice CIA model and Th17 cells. This project will elucidate the essential role of “SREBP/fatty acid metabolism/Th17 differentiation” axis in anti-RA effect of AFS and AC, and provide new insights into traditional Chinese medicine as drugs for the therapy of RA.

通过调节脂质代谢来抑制Th17细胞分化是治疗类风湿关节炎（RA）的重要策略。地乌总皂苷（AFS）具有良好的抗炎免疫抑制活性，已完成III期临床试验，有望开发成为治疗RA的中药5类新药，但是其作用机制尚不明确。前期工作中，我们发现AFS及其主要活性成分Anhuienosides C (AC) 能有效降低CIA小鼠脾脏Th17细胞比例；AFS和AC可抑制Th0细胞分化成Th17细胞，且能调控SREBP的转录及其介导的脂质代谢。在此基础上，本项目拟基于基因敲除的CIA小鼠模型及Th17细胞，采用免疫组化、报告基因、免疫印迹、Q-PCR及RNAi等技术，从动物、细胞、分子多层次研究并比较AFS及AC对SREBP介导的脂质代谢信号通路和Th17细胞分化相关蛋白的影响，阐明地乌皂苷通过“SREBP-脂质代谢-Th117细胞分化”轴抗RA的作用机制，为RA的中医药治疗提供科学依据。

本项目通过建立LPS诱导RAW264.7细胞释放TNF-α、IL-6的模型评价地乌皂苷类化合物的抗炎活性，得到了抗炎活性最好的化合物Anhuienoside C（AC）。并通过牛二型胶原诱导建立了类风湿性关节炎（CIA）小鼠模型研究地乌总皂苷(AFS) 和AC的抗类风湿关节炎活性，从巨噬细胞、T细胞活化以及Th17细胞分化等多角度阐明了AFS和AC抗类风湿关节炎的作用机制。研究结果显示：AFS和AC抗类风湿关节炎是一个多途径协助的复杂机制，但主要通过SREBP信号通路介导的脂质代谢引起Th17细胞极化受控，调节了IL-17炎症因子的释放，以及抑制Th1细胞释放IFN-γ而起到治疗类风湿性关节炎。