Roseburia intestinalis-derived inosine improves intestinal motility by activating TGF-β1/p-Smad3/Transgelin signaling axis.

BACKGROUND

Functional constipation (FC) is a prevalent gastrointestinal disorder marked by impaired intestinal motility, affecting millions worldwide and significantly diminishing quality of life. Current therapeutic strategies provide only transient symptom relief and fail to restore colonic motor function. This study aims to explore the causal role of gut microbiota in FC, focusing on the mechanisms by which Roseburia intestinalis (RI) and its metabolite inosine improve intestinal motility.

METHODS

To identify protective gut taxa, we first performed bidirectional Mendelian randomization (MR) integrated with 16S rRNA sequencing on fecal samples from 30 FC patients and 30 healthy controls. Loperamide-induced constipation mouse models evaluated RI gavage effects on motility indicators, including fecal output (frequency and water content), gastrointestinal transit time, intestinal propulsion rate, colonic bead expulsion time, and ex vivo organ bath contractions. To elucidate the underlying mechanism, we conducted RNA sequencing on colonic tissues. Furthermore, comparative metabolomics was employed to identify the key bacterial metabolites responsible for RI's effects. Finally, the proposed mechanism of action for both RI and its key metabolite was validated through a combination of histology, immunohistochemistry, immunofluorescence, quantitative PCR, and Western blot analyses.

RESULTS

MR and sequencing identified RI as a protective factor (OR = 0.8675, P < 0.05), with reduced abundance in FC patients negatively correlating with symptom severity and quality-of-life scores. In constipation mouse models, RI gavage promoted intestinal motility, enhanced fecal water content and gastrointestinal transit, and ameliorated mucosal injury while improving mucus secretion. RNA sequencing revealed upregulation of Tagln (transgelin) and enrichment of the TGF-β pathway after RI intervention. Metabolomics analysis pinpointed inosine as a key RI-derived small-molecule metabolite, enriched in RI supernatant and depleted in FC patient feces. Inosine gavage improved motility and histology, increased TGF-β1, p-Smad3, and transgelin, and these effects were abolished by the TGF-β receptor inhibitor SB431542.

CONCLUSIONS

These findings established that RI and its metabolite inosine promote intestinal motility via TGF-β1/p-Smad3 signaling and identified transgelin as the terminal effector of this cascade, offering mechanistic insight into microbiota-host interactions and pointing to therapeutic targets for gastrointestinal motility disorders.