Abstract
Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disorder characterized by epithelial barrier disruption, excessive immune activation and gut microbial dysbiosis. Dietary amino acids have emerged as potential regulators of intestinal homeostasis; however, whether L-proline (Pro), a functional amino acid involved in cellular metabolism and tissue repair, modulates intestinal inflammation through microbiota–host interactions remains unclear. In this study, we investigated the protective effects and underlying mechanisms of Pro supplementation in dextran sulfate sodium (DSS)-induced colitis. Pro administration significantly ameliorated colitis-associated phenotypes, including body weight loss, disease activity index elevation, colon shortening and histological damage. Furthermore, Pro improved intestinal barrier integrity by enhancing mucus secretion and restoring tight junction proteins, including Zonula Occludens-1 (ZO-1), Claudin-11 and Occludin. Transcriptomic analysis revealed that Pro supplementation induced extensive transcriptional remodeling and was associated with the regulation of inflammation-related pathways. Consistently, Pro modulated the expression of pro-inflammatory cytokines and attenuated activation of NF-κB/STAT3 signaling in colonic tissues. Gut microbiota profiling demonstrated that Pro reshaped DSS-induced microbial dysbiosis by enriching beneficial bacterial taxa, including Lactobacillus, Lachnospiraceae_NK4A136_group, Ruminococcus, Christensenellaceae_R-7_group and Odoribacter, while reducing inflammation-associated taxa such as Escherichia-Shigella. Importantly, fecal microbiota transplantation (FMT) experiments showed that microbiota from Pro-treated donors conferred protective effects against colitis in recipient mice, supporting the functional contribution of Pro-associated microbial remodeling to intestinal protection. Collectively, our findings reveal that Pro alleviates intestinal inflammation through coordinated regulation of gut microbiota composition, epithelial barrier integrity and inflammatory responses. This study provides new insights into the role of Pro in modulating microbiota–host interactions and highlights Pro as a potential nutritional strategy for maintaining intestinal health.