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This figure is drawn using Figdraw (https://www.figdraw.com/static/index.html#/). [Display omitted] • WP ameliorated DSS-induced colitis symptoms and alleviated intestinal barrier damage in UC mice. • WP suppresses inflammatory responses in UC mice by inhibiting macrophage M1 polarization and TLR4-NFκB signaling pathway. • WP was able to modulate the gut microbiota by increasing the abundance of the beneficial bacteria and decreasing the abundance of the harmful bacteria. The incidence of ulcerative colitis (UC) is increasing each year, and its course is characterized by alternating remission-relapse; currently, there is no effective treatment for UC. Peptides produced from the hydrolysis of walnut proteins have physiological functions such as anti-inflammatory and antioxidant properties. The aim of this study was to analyze the preventive effect of walnut polypeptide (WP) on UC and to preliminarily screen bioactive peptides in WP. The results of in vitro experiments showed that WP was able to alleviate the LPS-induced inflammatory response by inhibiting M1 polarization in Raw 264.7 cells and thereby alleviating the LPS-induced inflammatory response. The results of in vivo experiments showed that dietary supplementation with 500 mg/kg WP significantly alleviated dextran sodium sulfate (DSS)-induced weight loss, colon shortening, and elevated DAI scores. WP treatment was able to suppress DSS-induced inflammatory responses in mice by inhibiting macrophage M1 polarization and NF-κB signaling pathway. WP intervention also helped maintain intestinal barrier integrity by promoting tight junction protein expression and mucus secretion. In addition, 16S rDNA sequencing showed that WP ameliorated gut microbial disorder induced by DSS, as evidenced by a decrease in the abundance of harmful bacteria (Erysipelatoclostridium , Helicobacter , and Parasutterella) and an increase in the abundance of beneficial bacteria (Flavonifractor , Anaerotignum , and Alistipes) after WP intervention. In conclusion, this study demonstrates that WP ameliorates DSS-induced colonic inflammation in mice by inhibiting inflammation, modulating the gut microbiota and repairing the intestinal barrier. [ABSTRACT FROM AUTHOR]