Department of Pharmacology of Chinese Materia Medica, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China.
Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China.
Int Immunopharmacol. 2020 Dec;89(Pt A):107047. doi: 10.1016/j.intimp.2020.107047. Epub 2020 Oct 8.
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.
此前，我们曾报道口服积雪草苷（积雪草的主要活性三萜类化合物）可通过在小肠中诱导调节性 T（Treg）细胞的产生发挥抗关节炎作用。本研究旨在探讨积雪草苷诱导 Treg 细胞的作用部位和机制。在胶原诱导性关节炎（CIA）大鼠模型中，口服积雪草苷可显著缓解关节炎症状，但它的主要代谢产物积雪草酸则没有这种作用，提示积雪草苷以原型发挥作用。免疫组化和 qPCR 检测结果显示，积雪草苷可增加大鼠回肠而非十二指肠和空肠中 Treg 细胞的数量，并促进 Foxp3 和 IL-10 的表达。出乎意料的是，积雪草苷对体外 Treg 细胞分化以及 Foxp3 和 IL-10 的表达并无显著影响。广谱抗生素联合使用可显著降低 CIA 大鼠中积雪草苷的抗关节炎作用。16S rRNA 基因序列显示，积雪草苷可逆转关节炎状态下肠道微生物群的变化，并使厚壁菌门、丁酸球菌属、粪杆菌属、丁酸梭菌等多种细菌丰度增加。GC-MS 检测结果表明，积雪草苷可提高 CIA 大鼠盲肠内容物中乙酸和丁酸的水平，但不影响其他短链脂肪酸（SCFAs）的水平。丁酸而非乙酸可诱导 Treg 细胞的体外分化及 Foxp3 和 IL-10 的表达。因此，当积雪草苷与丁酸盐合酶的竞争性抑制剂庚酰辅酶 A 共同给药时，其对丁酸产生、Treg 细胞比例和关节炎的影响几乎消失。这些发现表明，口服积雪草苷以原型诱导 Treg 细胞的产生，从而发挥抗关节炎作用，而非代谢产物，回肠是主要作用部位。积雪草苷的作用机制可概括为：增加产丁酸菌的丰富度-上调肠道丁酸水平-诱导 Treg 细胞分化和 IL-10 表达。
