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Given the pivotal significance of the interactions between the gut microbiota and the host on governing bowel movement, we employed a multi-modal approach integrating animal experiments, ELISA, histopathology, qRT-PCR, GC-MS, and 16S rRNA metagenomics to evaluate the functional potential of Lactobacillus rhamnosus LRa05 against loperamide-induced constipation in mice. LRa05 treatment markedly alleviated constipation symptoms, as evidenced by reduced first black stool expulsion time, increased fecal moisture, and enhanced intestinal motility. Mechanistic investigations revealed that LRa05 balanced gastrointestinal regulatory peptides. It also downregulated aquaporin (AQP4\/AQP8) mRNA levels and activated the SCF\/C-Kit signaling pathway. These effects contributed to the restoration of intestinal peristalsis. Furthermore, LRa05 rebalanced gut microbiota composition by enriching beneficial, including Alloprevotella and Lachnospiraceae NK4A136, key SCFA producers. Thus, LRa05 could boost short chain fatty acid (SCFA) production, which is vital for stimulating intestinal motility, improving mucosal function, and relieving constipation. These findings demonstrated that LRa05 could mitigate constipation through a multi-target mechanism: regulating motility-related gene transcription, restructuring the microbial community, balancing gastrointestinal peptides, repairing the colonic mucosa, and promoting SCFAs for fecal hydration. Our study positions LRa05 as a promising probiotic candidate for constipation management.<\/jats:p>","DOI":"10.3390\/foods14132293","type":"journal-article","created":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T06:18:36Z","timestamp":1751264316000},"page":"2293","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Lactobacillus rhamnosus LRa05 Alleviates Constipation via Triaxial Modulation of Gut Motility, Microbiota Dynamics, and SCFA Metabolism"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-3111-5963","authenticated-orcid":false,"given":"Jingxin","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Food Nutrition and Safety, College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1697-235X","authenticated-orcid":false,"given":"Qian","family":"Li","sequence":"additional","affiliation":[{"name":"Nutritious and Healthy Food Sino-Thailand Joint Research Center, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shanshan","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Nutrition and Safety, College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Nutrition and Safety, College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Song","sequence":"additional","affiliation":[{"name":"Nutritious and Healthy Food Sino-Thailand Joint Research Center, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0629-8421","authenticated-orcid":false,"given":"Tao","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Nutrition and Safety, College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Nutrition and Safety, College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China"},{"name":"Nutritious and Healthy Food Sino-Thailand Joint Research Center, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1232","DOI":"10.1053\/j.gastro.2019.12.034","article-title":"Mechanisms, evaluation, and management of chronic constipation","volume":"158","author":"Bharucha","year":"2020","journal-title":"Gastroenterology"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Yuan, M., Zhang, Z., Liu, T., Feng, H., Liu, Y., and Chen, K. 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