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

Salmonella enterica 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 in silico and was verified by CETSA and DARTS assays in vitro. Limonin inhibited the enzyme activities and expression of SopB and SopE2 in vitro. 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 Salmonella-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 S. 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 (Mucin 1, Mucin 2, Occludin, Claudin-3 and ZO-1). A gut microbiota analysis revealed that limonin treatment influenced α- and β-diversity of the flora and increased the counts of the beneficial bacteria Muribaculum and Faecalibaculum 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.