Alfalfa Polysaccharide Alleviates Colitis by Regulating Intestinal Microbiota and the Intestinal Barrier Against the TLR4/MyD88/NF-κB Pathway

<b>Background/Objectives</b>: Ulcerative colitis (UC) pathogenesis involves gut barrier dysfunction, dysregulated immune responses, and gut microbiota imbalance. Alfalfa polysaccharide (APS), a bioactive compound with immunomodulatory potential, remains underexplored in intestinal inflammation. While APS exhibits anti-inflammatory properties in vitro, its in vivo efficacy, mechanisms, and ability to restore gut microbiota and barrier integrity in UC are unclear. This study aims to investigate the treatment effect of APS on dextran sulfate sodium (DSS)-induced colitis in mice and confirm its prebiotic potential. <b>Methods</b>: A mouse model of ulcerative colitis was induced by DSS. RNA sequencing, Western blotting, the terminal deoxynucleotidyl transferase dUTP nick end labeling technique, and an immuno-histochemical technique were used to study the mechanism of action by which APS at different dosages relieves DSS-induced colitis. <b>Results</b>: The findings show that APS alleviated the symptoms of colitis in mice given DSS, improved the gut morphology, heightened goblet cells production, increased the levels of IL-10 and IL-22, decreased the levels of TNF-α, IL-1β, and IL-6, and prevented the activation of the TLR4/MyD88/NF-κB pathways. Additionally, they maintained the integrity of the intestine by enhancing the expression of the mucins MUC2 and MUC5AC and by increasing the amounts of ZO-1, Occludin, and Claudin-1 proteins. Moreover, APS supported the growth of probiotic bacteria, including <i>unclassified_f_lachnospiraceae</i>, <i>Parabacteroides</i>, <i>Alistipes,</i> and <i>Mucispirillum</i>, and in particular, <i>Parabacteroides distasonis</i>, which is strongly associated with decreased pro-inflammatory cytokine through the inhibition of the TLR4-MyD88-NFκB pathways. <b>Conclusions</b>: APS can be used as a new type of prebiotic to improve UC by regulating intestinal flora and enhancing intestinal barrier function against the TLR4-MyD88-NFκB pathway.