基于肠道芳烃受体和黏膜免疫揭示异喹啉类生物碱抗类风湿关节炎的作用机制

Isoquinoline alkaloids are important repertoire for the discovery of anti-rheumatoid arthritis (RA) drugs. Among them, sinomenine, tetrandrine and norisoboldine are effective after oral administration, and the preparations of the first two have been successfully marketed. However, the plasma concentrations of the three alkaloids, orally administered, are far below the minimal effective concentrations in in vitro experiments. Their in vivo mechanisms of action remain unclear. Based on our latest understandings on the mechanisms of norisoboldine, this project studies the effects of the three alkaloids mentioned above on muscoal immune system (MIS) in RA model rats, and explores the internal relationship between regulation of MIS and anti-RA property in an attempt to clarify intestinal regulatory T cells (Tregs) as the target cells. The molecular mechanisms for these alkaloids promoting differentiation or activation of Tregs should be addressed. Moreover, the effects of alkaloids on intestinal aryl hydrocarbon receptor (AhR) and related signaling pathway are investigated. By using specific antagonists and RNA interference technique, we make sure that intestinal AhR is the key player for these alkaloids activating Tregs and therefore exhibiting anti-RA action. Overall, from "regulation of AhR-promotion of intestinal Treg differentiation/activation-inhibition of autoimmune response", this project provides a new perspective to elucidate shared anti-RA mechanisms of isoquinoline alkaloids. The findings obtained from this project should offer reasonable explanations for the clinical applications of sinomenine and tetrandrine, and provide evidence for further development of norisoboldine. This projct may also offer a paradigm for the studies of active ingredients from traditional Chinese medicines with similar pharmacokinetic characteristics to these alkaloids.

异喹啉类生物碱是抗类风湿关节炎（RA）药物发现的重要资源，其中口服有效的包括青藤碱、粉防己碱和去甲异波尔定，前两者的制剂已成功上市。但这些化合物的口服血药浓度与体外细胞试验的最低有效浓度相距甚远，其体内效应机制尚不清楚。本项目依据我们对去甲异波尔定作用机制的最新认识，在RA模型大鼠研究上述三个化合物对肠道黏膜免疫的调节作用及其与抗关节炎活性的内在联系，确定肠道Treg为靶细胞，阐明其促进Treg分化或活化的分子机制。此外，观察化合物对肠道芳烃受体及其信号途径的影响，采用拮抗剂和RNA干扰等手段，证实该受体是化合物激活Treg发挥抗关节炎效应的关键中介。从"调节芳烃受体-促进肠道Treg分化/活化-抑制自身免疫应答"的全新视角，探索异喹啉生物碱抗RA作用的共性规律。为青藤碱和粉防己碱的临床应用提供合理解释，为去甲异波尔定的开发利用提供依据，同时为具有类似药动学特点中药有效成分的研究提供示范。

异喹啉类生物碱是抗类风湿关节炎（RA）药物发现的重要资源，其中口服有效的包括青藤碱、粉防己碱和去甲异波尔定，前两者的制剂已成功上市。但这些化合物的口服血药浓度与体外细胞试验的最低有效浓度相距甚远，其体内效应机制尚不清楚。本项目在RA模型动物和相关细胞研究上述三种生物碱对肠道黏膜免疫的调节作用及其与抗关节炎活性的内在联系，明确了肠道Treg细胞为其靶细胞，揭示了异喹啉生物碱作用的共性规律。主要研究内容和结果如下：. 1. 异喹啉生物碱在关节炎大鼠的药动学行为有别于在正常大鼠：青藤碱在CIA大鼠、去甲异波尔定在AIA大鼠治疗给药，其药动学参数有别于在正常大鼠，主要表现为Cmax和AUC的升高。究其原因，关节炎模型大鼠的肠道P-糖蛋白表达和活力均下降，而这些生物碱为P-糖蛋白底物。然而，值得注意的是，即使在关节炎大鼠，生物碱的Cmax仍远远低于其体外试验的最低有效浓度。. 2. 肠道为生物碱的初始作用部位：基于上述药动学研究，结合肠道外给药试验，发现肠道为三种生物碱的初始作用部位，为其药效学—药动学不相关现象提供了合理的解释。. 3. 三种生物碱经由芳烃受体（AhR），纠偏Th17/Treg失衡状态：在AIA、CIA大鼠和CIA小鼠，结合体外T细胞分化和功能试验，明确这些生物碱通过激活AhR，下调Th17细胞，明显上调Treg细胞，逆转关节炎动物的Th17/Treg失衡状态。. 4. 三种生物碱具有类似的抗关节炎作用机制：在关节炎模型动物，通过采用AhR阻断剂，明确了生物碱的机制为：激活肠道AhR—促进肠道Treg细胞分化/活化—抑制全身性自身免疫应答—改善关节炎。. 上述工作从全新的视角诠释了三种异喹啉生物碱的抗关节机制，为其PK-PD不相关现象提供了合理的解释。为青藤碱、粉防己碱的临床应用和去甲异波尔定的深入研究开发提供了依据，同时为具有类似药动学特点的中药有效成分作用机制的研究提供了示范。