Alleviation of Loperamide-Induced Constipation by Lactiplantibacillus plantarum SYS1: Role of Properistaltic Metabolites and Promotion of Enteric Nerve Development via 5-HT4R Activation

Lactiplantibacillus plantarum SYS1 was identified through in vitro probiotic screening and zebrafish models as a potential candidate for alleviating constipation. Further studies confirmed its efficacy in constipated mice, evidenced not only by phenotypic indicators but also by improvements in intestinal villi morphology, expression of gut barrier-related genes and aquaporin genes, and beneficial modulation of gut microbiota structure. Colonic transcriptomics, qPCR, and Western blot results identified the serotonin receptor 4 (5-HT4R) as the likely primary target for SYS1's anti-constipation effect. Subsequent 5-HT4R antagonism experiments confirmed this, as SYS1 lost its efficacy when 5-HT4R was blocked. Further characterization of the enteric nervous structure revealed that SYS1 partially reversed loperamide - induced enteric nervous degeneration, an effect also dependent on 5-HT4R. Experiments in zebrafish and mice showed that the fermented supernatant of SYS1 could alleviate constipation, also primarily targeting 5-HT4R. Non-targeted metabolomics identified maleic acid as a characteristic metabolite of SYS1. Both zebrafish and mouse models demonstrated the anti-constipation effect of maleic acid. This study reveals that L. plantarum SYS1 alleviates constipation by promoting enteric neurogenesis through its metabolite, which activates the 5-HT4R.