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.