Esculetin Combats Multidrug-Resistant Salmonella Infection and Ameliorates Intestinal Dysfunction via the Nrf2 Pathway

Abstract

The increasing incidence of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Tm), known for causing invasive enteric infections, presents a significant public health challenge. Given the diminishing efficacy of existing antibiotics, it is imperative to explore novel alternatives for the treatment of MDR S. Tm infections. Here, we identified esculetin (EST), a natural coumarin abundant in dietary foods and herbs, as a compound exhibiting broad-spectrum antibacterial properties against a range of MDR bacteria. Our findings demonstrate that EST effectively inhibited the proliferation and expansion of MDR S. Tm in both in vitro experiments and animal models. Specifically, EST significantly downregulated the type 3 secretion system-1 (T3SS-1) virulence expression of MDR S. Tm, thereby preventing its invasion into intestinal epithelial cells. In S. Tm-infected mice, we observed cecal injury characterized by the upregulation of inflammatory cytokines, a reduction in goblet cell numbers, a decreased expression of tight junction proteins, and microbial dysbiosis. Conversely, EST treatment ameliorated these pathological changes induced by S. Tm infection and reduced oxidative stress by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, thereby improving intestinal barrier function. These results suggest that dietary coumarins or a targeted plant-based diet may offer a promising strategy to counteract MDR bacteria-induced enteric diseases.

Keywords: Nrf2; S. Tm; coumarin; esculetin; intestinal barrier; multidrug-resistant (MDR).

Figure 1

Sources and structures of coumarins. The numbers 1 to 8 indicate the positions of carbon atoms in the chemical structure.

Figure 2

Antibacterial activities of EST on diverse pathogenic bacteria. (A) Growth curves of S. Tm, E. coli, and K. pneumonia with EST supplementation. (B) Growth curves of MRSA, S. chromogenes, and B. velezensis with EST supplementation.

Figure 3

EST reduces the invasion of S. Tm into intestinal epithelial cells. (A) Confocal images showing internalized S. Tm 15E475 (green) in IEC-6 cells. The 3D images were acquired using confocal laser scanning microscopy (CLSM), where F-actin was visualized with rhodamine phalloidin (red) and nuclei with DAPI (blue). (B) The number of internalized S. Tm 15E475 in IEC-6 cells with or without EST treatment. (C) Relative expression of virulence genes (VGs) of S. Tm 15E475 with or without EST treatment. Results represent the mean ± standard error. p values were calculated using independent-samples t-tests. ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

EST decreases S. Tm loads and disease severity in infected mice. (A) Experimental design for the establishment of S. Tm-infected and EST-treated mouse models. (B) S. Tm shedding in feces of mice for 96 h. (C) Representative H&E staining images of the cecum, liver, and spleen in uninfected, S. Tm-infected, and EST-treated mice. (D) S. Tm loads in the jejunum, ileum, cecum, and colon. (E) S. Tm loads in the liver and spleen. (F) Inflammatory cytokine levels in the cecum. Results represent the mean ± standard error. p values were calculated using independent-samples t-tests (D,E) or one-way ANOVA followed by Fisher’s LSD post hoc tests (F). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

EST improves the microbial barrier of S. Tm-infected mice. (A,B) α-diversity and β-diversity analyses. (A) α-diversity analysis (Shannon and Chao1 indexes) of cecal microbiota richness and diversity. (B) Principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS) score plot based on the binary Jaccard distance metrics, with each symbol representing one sample. (C) Heatmap analysis illustrating changes in relative abundance of the top 30 bacterial species of the cecal microbiota. (D) The cladogram displays taxonomic tree of differentially abundant taxa. (E,F) Relative abundance of key genera in the cecal microbiota based on the LefSe results. Solid and dashed lines indicate the mean and median, respectively. Results represent the mean ± standard error. p-values were calculated using the independent-samples t-test. ** p ≤ 0.01; *** p ≤ 0.001.

Figure 6

EST enhances the physical intestinal barrier against S. Tm infection. (A–D) Images of PAS-stained sections and the quantification of goblet cells in the cecum (A,B) and colon (C,D). (E) Expression of Claudin-1, ZO-1, and E-cadherin protein in the cecum. Protein levels were normalized relative to β-actin (n = 6). Results represent the mean ± standard error. p values were calculated using one-way ANOVA followed by Fisher’s LSD post hoc test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 7

EST ameliorates S. Tm-induced intestinal oxidative injury via activating the Nrf2 pathway. (A) Concentrations of antioxidant enzymes (GPx, CAT and SOD), T-AOC and MDA in the cecum (n = 6). (B,C) Cecal iNOS (B), Keap1 and p-Nrf2 (C) protein expression in uninfected, S. Tm and EST-treated groups. Relative protein levels were normalized to β-actin (n = 6). Results represent the mean ± standard error of the mean. p-values were calculated using one-way ANOVA followed by Fisher’s LSD post hoc tests. * p ≤ 0.05; *** p ≤ 0.001; ns, not significant.