基于芳烃受体和Th17/Treg平衡探索草豆蔻主要有效成分抑制溃疡性结肠炎的作用机制

Aryl hydrocarbon receptor (AhR) and the balance of Th17/Treg cells play important roles in the development and progression of ulcerative colitis (UC). Semen Alpiniae Katsumadai (SAK), a traditional Chinese medicine, possesses the ability to strengthen spleen and eliminate pathogen dampness, and can be used to treat intestinal diseases due to spleen asthenia and invasion of pathogen dampness. Previously, we demonstrated that the total flavonoids were the main active fraction of SAK for alleviating UC, and the mechanisms may involve the regulation of Th17/Treg balance and activation of AhR. In this study, we identify the key active constituent of the total flavonoids by comparing the inhibitory effects of various flavonoid constituents on DSS-induced UC in mice. Based on the effects on the differentiation and function of Th17 and Treg cells, the mechanisms and target cell for this active constituent regulating T lymphocyte imbalance can be elucidated. Moreover, the enhancing effects of this active constituent on expression and activation of AhR in Th17 or/and Treg cells are observed by in vivo and in vitro studies, and the relative signal pathways are explored. The close relationship between its activation of AhR and regulation of T lymphocyte balance and consequent anti-UC action should be confirmed by using specific antagonists and RNA interference technique. In view of “activating AhR-improving the imbalance of Th17/Treg cells-inhibiting the immune inflammation”, the underlying mechanisms of the main active constituent in SAK can be elucidated. The findings can provide evidence for the further development of this constituent as an anti-UC drug, and offer a paradigm for the recognization of anti-UC mechanisms of the flavonoid constituents occurring in traditional Chinese medicines.

芳烃受体和Th17/Treg平衡与溃疡性结肠炎（UC）的发生发展密切相关，中药草豆蔻具有健脾去湿之功效，可治疗UC等脾虚并湿邪入侵所致的肠道疾病。申请人前期研究发现，总黄酮系草豆蔻改善UC的有效部位，其作用可能与调节Th17/Treg平衡及活化芳烃受体相关。本课题以DSS诱导小鼠UC，比较研究草豆蔻主要黄酮成分的改善作用，明确主要有效成分。考察其对Th17、Treg细胞分化和功能的影响，探明其调节T细胞失衡的机制和效应细胞。体内外研究该成分对Th17或Treg细胞中芳烃受体表达和/或活化的促进作用，揭示相关的信号途径。采用受体阻断剂和RNA干扰等手段，证实芳烃受体是该成分调节T细胞平衡、发挥抗UC作用的关键中介。即从“激活芳烃受体—改善Th17/Treg失衡—抑制免疫性炎症”的角度，阐明草豆蔻主要有效成分抑制UC的机制，为其开发应用打下基础，同时为中药黄酮类成分抗UC机制的研究提供借鉴。

溃疡性结肠炎（UC）的发生发展与Th17/Treg平衡及芳烃受体（AhR）活化均密切相关，且中药草豆蔻可治疗UC等肠道疾病。申请人前期研究结果指出总黄酮为草豆蔻抗UC的有效部位，机制可能与调节Th17/Treg平衡及AhR相关。本课题主要比较研究草豆蔻主要黄酮成分对UC的改善作用，探明其效应物质基础及关键作用机制。.研究发现，1）山姜素、豆蔻明和乔松素为草豆蔻总黄酮包含的主要成分，山姜素（30 mg/kg）和豆蔻明（30 mg/kg）对小鼠结肠炎呈现显著的抑制效应，山姜素作用优于豆蔻明，而乔松素（30 mg/kg）作用不明显。结合前期结果，提示山姜素系草豆蔻改善UC的主要有效成分；2）山姜素（7.5, 15, 30 mg/kg）剂量依赖性改善结肠炎小鼠疾病症状，调节肠系膜淋巴结（MLNs）和结肠粘膜固有层（LPs）Th17和Treg细胞的比率。体外研究中，山姜素直接诱生Treg细胞，但对Th17细胞分化无明显的影响。提示，恢复Th17/Treg平衡为山姜素发挥抗UC作用的关键机制，Treg为其作用的靶细胞；3）山姜素可进入T细胞，促进AhR/HSP90解离、AhR转核和AhR/ARNT形成，上调AhR依赖的荧光素酶报告基因活力，升高CYP1A1水平和活力，呈现明显的AhR激活作用；4）深入研究发现，山姜素通过激活T细胞中AhR，选择性促进miR-302表达，抑制DNMT-1表达，降低Foxp3启动子区域的甲基化程度，促进CREB与Foxp3甲基化序列的结合，发挥诱生Treg细胞作用；5）体内联合应用山姜素和AhR拮抗剂，拮抗剂几乎完全抵消山姜素对小鼠结肠炎的改善作用，对MLNs和LPs中Treg细胞比例的上调，对miR-302/DNMT-1/CREB/Foxp3启动子区域甲基化程度的调控，提示AhR为山姜素抗UC的靶蛋白；6）此外，研究成果亦证实另一活性成分豆蔻明通过激活AhR，干预Nrf2/NQO1/ROS信号，抑制NLRP3炎症小体活化，发挥抗UC作用；7）研究成果已发表SCI论文2篇，1篇IF=5.638；此外，在审1篇，IF=5.595；论文水平超预期。.综上，本研究主要证实“激活AhR调节Th17/Treg失衡改善免疫性炎症”为草豆蔻主要有效成分山姜素改善UC的主要效应机制，促进其进一步研究与开发，并为中药黄酮类成分抗UC作用机制的研究提供借鉴。