基于“PPARγ—脂肪酸氧化—Th17/Treg平衡”途径揭示桑枝抗类风湿关节炎效应成分桑色素的作用机制

The imbalance of Th17/Treg cells plays a crucial role in development and progression of rheumatoid arthritis (RA). Fatty acid oxidation and its upstream protein PPARγ constitute the regulatory axis for the differentiation and phenotypic transformation of Th17 and Treg cells. Ramulus Mori, a traditional Chinese medicine, possesses the abilities to dispel wind and remove obstruction in meridians of joints, and is used to treat Arthromyodynia (Bi Zheng) including RA for a long history. In this study, we identify the regulatory effect of morin, the main active ingredient of Ramulus Mori, on the imbalance of Th17/Treg cells in mice with collagen-induced arthritis, and elucidate the target cell by investigating the regulation of differentiation, phenotypic transformation and activation of Th17 and Treg cells via in vitro and in vivo experiments. Then, the regulatory effect of morin on fatty acid oxidation and underlying mechanism will be explored. By using specific inhibitors and RNA interference technique, the close relationship between the regulation of fatty acid oxidation and the restoration of Th17/Treg balance should be verified. In addition, PPARγ is confirmed as a potential target protein of morin by using affinity assay and transcriptional activation analysis. In view of “activating PPARγ-regulating fatty acid oxidation-restoring Th17/Treg balance”, the underlying mechanisms for anti-RA effect of morin can be elucidated. The findings can provide evidence for the clinical application of Ramulus Mori and the further development of morin as an anti-RA drug, and offer a paradigm for the recognization of anti-RA mechanisms of the flavonoid ingredients occurring in traditional Chinese medicines.

Th17/Treg失衡与类风湿关节炎的发生发展紧密相关，而脂肪酸氧化及其上游PPARγ的激活是Th17、Treg细胞分化和表型转化的调节枢纽。桑枝具有祛风湿、利关节之功效，为治疗类风关等“痹症”的常用中药，桑色素为其主要活性成分。本课题考察桑色素灌胃给药对胶原关节炎小鼠Th17/Treg失衡的调节作用，体内外研究桑色素对Th17和Treg细胞分化、转化及活化的影响，明确其主要效应细胞。在此基础上，探讨桑色素对脂肪酸氧化代谢的调节作用和机制，采用阻断剂和RNA干扰等手段验证其在调节Th17/Treg平衡中的关键作用。此外，通过亲和力测定和转录激活分析等手段，证实PPARγ是桑色素的潜在靶蛋白。即从“激动PPARγ-调控脂肪酸氧化-恢复Th17/Treg平衡”的角度，阐明桑色素抗类风关的分子机制，为桑枝的临床应用及桑色素的开发利用提供理论依据，同时为中药黄酮类成分抗类风关机制的研究提供借鉴。

本项目综合考察桑色素对Th17/Treg平衡的调节作用及其与抗关节炎活性的内在联系，明确“PPARγ-脂肪酸代谢”为其关键作用途径。结果总结如下：1. 抗关节炎作用：在多种关节炎动物模型上，证实桑色素单独使用具有良好的抑制作用，下调关节炎指数、血清炎性介质及IgG类抗体水平，改善关节滑膜部位炎症、血管新生和成纤维细胞迁移，减轻骨损伤。然而，桑色素与双氯芬酸钠联用时，后者的代谢被加速，体内暴露量降低，存在相互作用风险。2. 恢复Th17/Treg平衡：桑色素明显升高关节炎模型动物脾脏和淋巴结中Treg细胞比例，降低Th17细胞比例，同等程度调节两类细胞特异性转录和功能相关因子水平。体外实验研究结果亦证实，桑色素具有诱生Treg细胞，阻遏Th17细胞形成，并促进Th17细胞朝向Treg细胞进行表型转化的作用。3. 调控脂肪酸代谢：在Treg细胞分化条件下，桑色素促进CD36和CPT1的表达，加速脂肪酸摄取，上调呼吸速率，升高线粒体膜电位水平，促进脂肪酸氧化进程。随后，抑制磷酸果糖激酶PFK的活性，上调UDP-GlcNAc水平，增强TβRII和IL-2Rα（CD25）的N-糖基化修饰和信号转导，促进Treg细胞特异性转录因子Foxp3的合成。此外，在Th17细胞分化条件下，桑色素抑制FASN的转录，下调胞内脂质、甘油三酯和油酸水平，即阻遏脂肪酸合成进程。随后，借助油酸水平的下调，削弱RORγt与il-17a基因启动子和CNS2区的结合。4. 靶向PPARγ：桑色素能够直接与PPARγ的配体结合区域结合，激动PPARγ，进而调控FASN、CD36和CPT1的转录，干预Th17和Treg细胞的分化过程。PPARγ特异性拮抗剂GW9662或敲除PPARγ逆转桑色素对脂肪酸代谢、Th17和Treg细胞分化及关节炎疾病症状的调节。5. 课题研究成果已发表SCI论文3篇（IF1, 2=5.914, IF3=1.908），接收1篇（IF4=5.712），在投1篇（IF5=6.691）。另外，发表核心期刊论文1篇，综述论文1篇和会议论文1篇，水平超预期。.综上，本项目的研究成果证实“激活PPARγ-调节Th17/Treg失衡-改善免疫性炎症”为桑枝主要活性成分桑色素抗类风关的重要效应机制，促进其进一步研究与开发，并为中药黄酮类成分作用机制的研究提供借鉴。.