基于视黄酸对CD103+DC功能的调控作用揭示白背叶楤木总皂苷抗溃疡性结肠炎的机制

The leaf buds of Aralia chinensis L. var. nuda Nakai are used medicinally for enteritis and dysentery treatment in folks, of which the major active fraction is total saponions (TS). We have previously demonstrated that TS of A. chinensis leaf buds could significantly attenuate ulcerative colitis (UC) induced by DSS, regulated the expression of the specific transcription factors of Th17 and Treg, and enhanced the expression of ALDH in colon tissues in mouse. CD103+DC is the major cell type that highly expressing the key enzyme in retinoic acid production, ALDH. Furthermore, CD103+DC possesses potential efficacy in inducing Th17/Treg balance and maintain the immunologic homeostasis in the intestine through retinoic acid. In addition, in UC state, the functional derangement of CD103+DC drives the excessive immune response and tissue inflammation. In this project, we raise the hypothesis that “TS improve CD103+DC ALDH –ameliorate CD103+DC functional derangement – inhibit UC”, explore the effects and mechanisms of the management of TS on the functional derangement of colonic CD103+DC and also the effects and mechanisms on the colonic CD103+DC ALDH expressing and activity, in vivo, ex vivo and in vitro. Then utilizing specific inhibitor of ALDH, reveal the correlation between the improvement of CD103+DC ALDH expressing and activity, CD103+DC functional derangement amelioration and anti-ulcerative colitis effect, and then elucidate the action mode in anti-UC effect of TS, lay the foundation for the further development of major bioactive fraction of A. chinensis leaf buds, and shed light on the study the anti-UC with major bioactive component in traditional Chinese medicine.

白背叶楤木嫩叶芽苞在民间常用于治疗肠炎、痢疾，总皂苷为其主要活性部位。我们的前期研究发现，该总皂苷口服显著改善小鼠实验性溃疡性结肠炎（UC），调节结肠组织Th17和Treg特异性转录因子的表达，升高醛脱氢酶（ALDH）的表达。CD103+DC是结肠高表达ALDH的主要细胞，通过ALDH催化生成视黄酸，在诱导Th17/Treg平衡、维持肠道免疫稳态中发挥关键作用。CD103+DC功能紊乱是UC肠道异常免疫应答的驱动因子，因此，本课题提出“白背叶楤木总皂苷干预视黄酸生成—调控CD103+DCs功能—治疗UC”的工作假说，在UC模型，探索其对结肠CD103+DC功能紊乱的调控及机制；研究其上调ALDH表达的作用及机制；采用酶抑制剂，揭示其上调ALDH表达与调控CD103+DC功能及抗UC作用的关联，最终阐明该总皂苷抗UC的效应模式，为其开发利用打下基础，为中药有效成分抗UC机制研究提供示范。

溃疡性结肠炎（ulcerative colitis, UC）又称慢性非特异性溃疡性结肠炎，以腹痛、腹泻及黏液脓血便等为主要临床表现。UC病变多从直肠开始，逆向发展累及全结肠甚至末端回肠，病灶呈连续性弥漫性分布，病情反复，迁延不愈且有癌变倾向，被世界卫生组织列为现代难治病之一。UC在北美和北欧的患病率为全球最高，随着经济发展和生活方式日益西化，我国的UC患病率呈逐年增高趋势。同时，UC在中国人群中的临床表现有异于西方人群，且由于多种原因导致确诊较晚、确诊时病情更严重，为UC的治疗带来更大的挑战。传统医药对UC有独到的认识和丰富的诊疗实践，积累了大量理法方药，从中开发、寻找治疗UC的药物，具有重要的社会意义和实用价值。.本研究以云南少数民族用于治疗肠炎、痢疾的白背叶楤木嫩叶芽苞为研究对象，首先在葡聚糖硫酸钠诱导的小鼠实验性UC模型，证实其对小鼠一般体征、疾病活动指数及结肠组织病理学变化的显著改善作用。接着从免疫调节角度，发现总皂苷可有效恢复Th17/Treg平衡，表现为下调UC小鼠结肠组织中Th17的数量，上调Treg的数量，下调Th17细胞因子IL-17A含量及特异转录因子RORγt表达水平，上调Treg细胞因子TGF-β及特异转录因子FOXP3表达水平。总皂苷可显著下调结肠黏膜固有层树突状细胞（dendritic cell, DC）活化水平，下调其诱导Th0细胞分化为Th17细胞的能力，上调其诱导Th0细胞分化为Treg细胞的能力。同时，总皂苷可明显上调小鼠不同组织视黄酸的含量，并在体内外上调肠系膜淋巴结DC中视黄酸含量。进一步研究发现，总皂苷对视黄酸含量的上调作用与其上调视黄酸代谢关键酶ALDH1a1表达量相关。上述结果提示，总皂苷对小鼠UC的治疗作用，可能与上调视黄酸代谢关键酶ALDH1a1的表达，提高DC中视黄酸的水平，进而下调DC成熟程度，使DC倾向于诱导Treg，恢复Th17/Treg平衡有关。为证实这一关联，在DSS诱导的UC模型同时引入ALDH1a1抑制剂柠檬醛，结果发现，给予柠檬醛后，总皂苷对UC的改善作用被明显减弱，证实“干预视黄酸生成—调控DCs功能—调节Th17/Treg平衡—治疗UC”是白背叶楤木总皂苷发挥抗UC作用的途径之一。