Leven PRO-CR mediates 5-HT and gut microbiota via TPH1 to improve slow-transit constipation

Slow-transit constipation (STC) is a chronic disorder causing prolonged colonic transit and discomfort; this study explores the therapeutic potential of probiotics mixture (Leven PRO-CR) in modulating gut microbiota and enhancing intestinal motility. A loperamide-induced STC mouse model was established, and the effects of Leven PRO-CR treatment were evaluated by measuring defecation frequency, stool water content, and intestinal transit rate. Histopathological analyses, including Hematoxylin & Eosin (H&E) staining, immunohistochemistry (IHC), and TUNEL assay, were performed to assess colonic tissue integrity, interstitial cells of Cajal (ICCs) density, and apoptosis. The expression levels of serotonin (5-HT)-related markers (TPH1, 5-HT receptors, SERT) and enteric neural markers (NOS1, BDNF, TRPV1, GDNF) were analyzed using quantitative PCR (qPCR), Western blotting, and immunofluorescence staining. The gut microbiota composition was examined through 16S rDNA sequencing. Statistical analyses were conducted using GraphPad Prism, with significance set at P < 0.05. Leven PRO-CR significantly improved STC symptoms by reducing first defecation time, increasing stool frequency, fecal water content, and intestinal transit rate. We found that Leven PRO-CR restored colonic tissue integrity, enhanced ICC survival by upregulating c-Kit/SCF signaling, and reduced apoptosis. Leven PRO-CR upregulated TPH1 expression, increased 5-HT levels, and inhibited its degradation, thereby promoting intestinal motility. Leven PRO-CR also modulated NOS1, BDNF, TRPV1, and GDNF mRNA and protein expression, suggesting enhanced enteric nervous system function. 16S rDNA sequencing revealed increased microbial diversity and a restored balance of beneficial bacteria after Leven PRO-CR treatment, indicating the beneficial effects of Leven PRO-CR on balancing gut microbiota in STC. Overall, Leven PRO-CR alleviated slow-transit constipation potentially by enhancing 5-HT secretion, upregulating TPH1 channels, and restoring microbiota balance.