Dietary limonin alleviates <i>Salmonella</i> Typhimurium-induced colitis <i>via</i> dual targeting virulence SopB and SopE2 and inhibiting RAC1/CDC42/Arp2/3 pathway and regulating gut microbiota

<i>Salmonella enterica</i> serovar Typhimurium (STM) causes severe colitis, necessitating the development of effective drugs. Here, the dockings of limonin with the STM T3SS-1 virulence factor SopB or SopE2 showed strong binding activity <i>in silico</i> and was verified by CETSA and DARTS assays <i>in vitro</i>. Limonin inhibited the enzyme activities and expression of SopB and SopE2 <i>in vitro</i>. Furthermore, we found that limonin treatment significantly reduced the number of STM colony-forming units (CFUs) in infected HeLa and Raw264.7 cells, which resulted in a decrease in the rate of membrane ruffling mediated by SopB-regulated Arf6/Cyth2/Arf1-, RAC1-, and CDC42-driven Arp2/3-dependent actin polymerization and the SopE2-regulated CDC42/Arp2/3 pathway, and the confocal laser scanning microscopy analysis revealed that limonin treatment repressed the recruitment of the <i>Salmonella</i>-containing vacuole (SCV) biomarkers LC3, Rab7, GAL8 and NDP52. Furthermore, limonin treatment ameliorated STM-induced colitis by reducing the disease activity index (DAI), colon shortening, and MPO and EPO activities; mitigating the severity of <i>S</i>. Typhimurium-induced colitis damage; and influencing the levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α and IFN-γ) while increasing the levels of colonic epithelial barrier and tight junction genes (<i>Mucin 1</i>, <i>Mucin 2</i>, <i>Occludin</i>, <i>Claudin-3</i> and <i>ZO-1</i>). A gut microbiota analysis revealed that limonin treatment influenced α- and β-diversity of the flora and increased the counts of the beneficial bacteria <i>Muribaculum</i> and <i>Faecalibaculum</i> to regulate gut microbiota dysbiosis. Finally, colon SCFA measurements revealed that limonin treatment significantly increased acetate, butyrate, propionate and valerate concentrations. Thus, this study is an important reference for the anti-STM effects of limonin on induced colitis.