Previously, we reported that oral administration of madecassoside, the main active triterpene in Centella asiatica L., exerted anti-arthritis effect by inducing the generation of regulatory T (Treg) cells in small intestine. This study investigates the action site and mechanism of madecassoside to induce Treg cells. In collagen-induced arthritis (CIA) of rats, oral administration of madecassoside significantly alleviated arthritis symptoms, but its main metabolite madecassic acid did not, suggesting that madecassoside functions in the parent form. Madecassoside was shown to increase the number of Treg cells and promote the expression of Foxp3 and IL-10 in rat ileum rather than duodenum and jejunum, as detected using the immunohistochemistry assay and quantitative PCR assay, respectively. Unexpectedly, madecassoside was absent of significant effect on in vitro Treg cell differentiation and the expression of Foxp3 and IL-10. A combined use of broad-spectrum antibiotics resulted in significant reduction of the anti-arthritis effect of madecassoside in CIA rats. The 16S rRNA gene sequence showed that madecassoside could reverse the changes of gut microbiota under arthritis condition, and enrich several bacteria such as Lachnospiraceae, Butyricicoccus, Faecalibacterium, Butyricicoccus pullicaecorum and so on. GC-MS assay showed that madecassoside elevated the levels of acetic acid and butyric acid, but not other short chain fatty acids (SCFAs) in the cecum contents of CIA rats. Butyric acid rather than acetic acid could induce the in vitro differentiation of Treg cells and the expression of Foxp3 and IL-10. Accordingly, when madecassoside was co-administered with heptanoyl CoA, the competitive inhibitor of butyrate synthase, its effect on butyric acid production, Treg cell proportion and arthritis nearly disappeared. These findings indicate that oral madecassoside induces the generation of Treg cells and therefore displays anti-arthritis effect in the parent form but not metabolites, and the ileum is the main action site. The mechanism of madecassoside can be summarized as: expansion of the richness of butyrate-producing bacteria-up-regulation of intestinal butyrate level-induction of Treg cell differentiation and IL-10 expression.

先前，我们报道口服积雪草苷，积雪草中的主要活性三萜L.，通过诱导小肠中调节性T（Treg）细胞的产生而发挥抗关节炎作用。这项研究调查了马德卡西苷诱导Treg细胞的作用部位和机制。在大鼠的胶原蛋白诱发的关节炎（CIA）中，口服马德卡西苷可显着缓解关节炎症状，但其主要代谢产物马卡死酸却没有，这表明马德卡西苷以母体形式起作用。分别使用免疫组织化学测定和定量PCR测定法显示，马德卡索苷可增加大鼠回肠中Treg细胞的数量并促进Foxp3和IL-10的表达，而不是十二指肠和空肠。出乎意料的是，马德卡索甙对体外没有显着影响Treg细胞分化以及Foxp3和IL-10的表达。广谱抗生素的联合使用可显着降低马卡德西苷在CIA大鼠中的抗关节炎作用。16S rRNA基因序列表明，马卡德可甙可以逆转关节炎条件下肠道菌群的变化，并富集多种细菌，如旋毛虫科，丁酸球菌，费氏杆菌，普氏丁酸杆菌等。GC-MS分析表明，马卡德卡西甙可提高CIA大鼠盲肠中盲肠中乙酸和丁酸的含量，但不增加其他短链脂肪酸（SCFA）的含量。丁酸而不是乙酸可以诱导体外Treg细胞的分化以及Foxp3和IL-10的表达 因此，当马卡德花苷与庚酸合酶的竞争性抑制剂庚酰辅酶A并用时，其对丁酸产生，Treg细胞比例和关节炎的作用几乎消失了。这些发现表明，口服马卡德西甙可诱导Treg细胞生成，因此以母体形式显示抗关节炎作用，但不显示代谢产物，回肠是主要的作用部位。羟基积雪草苷的机理可归纳为：扩大产生丁酸的细菌的丰富度，上调肠丁酸水平，诱导Treg细胞分化和IL-10表达。