The Role of Dihydroresveratrol in Enhancing the Synergistic Effect of Ligilactobacillus salivarius Li01 and Resveratrol in Ameliorating Colitis in Mice

Abstract

Currently approved therapeutical strategies for inflammatory bowel diseases (IBD) suffer from variable efficacy and association with risk of serious side effects. Therefore, efforts have been made in searching for alternative therapeutics strategies utilizing gut microbiota manipulation. In this study, we show that the probiotic strain Ligilactobacillus salivarius Li01 (Li01) and the phytochemical prebiotic resveratrol (RSV) have synergistic effect in ameliorating colitis in mice. Oral coadministration of Li01 (10⁹ CFU/d) and RSV (1.5 g/kg/d) promoted restoration of various inflammatory injuries and gut microbiota composition, exhibiting a favorable anti-inflammatory effect in DSS-induced colitis mice. The combination treatment was associated with reductions in the levels of proinflammatory cytokines IL-1β and IL-6 and increases in the levels of the anti-inflammatory cytokine IL-17A in mouse serum. Moreover, the combination treatment was found to alter the composition and metabolism of the gut microbiota, especially influencing the production of short chain fatty acids and anti-inflammatory related molecules. The mechanism underlying the improved anti-inflammatory effect from the RSV and Li01 combination treatment was found to be associated with the environmental sensor mammalian aryl hydrocarbon receptor (AHR) and tryptophan metabolism pathway. Administration of RSV in combination with Li01 in different mouse model led to enhanced conversion of RSV into metabolites, including dihydroresveratrol (DHR), resveratrol-sulfate, and resveratrol-glucuronide. DHR was found to be the dominant metabolite of RSV in conventional and colitis mice. An increased DHR/RSV ratio was confirmed to activate AHR and contribute to an enhanced anti-inflammatory effect. DHR is considered as a potential AHR ligand. The DHR/RSV ratio also affected the serotonin pathway by controlling the expression of Tph1, SERT, and 5-HT₇R leading to amelioration of colitis in mice. Our data suggest that treatment with a combination of Li01 and RSV has potential as a therapeutic strategy for IBD; further investigation of this combination in clinical settings is warranted.

Figure 1

Therapeutic effect of RSV, Li01, and RSV+Li01 on DSS-induced colitis in mice. (a) Chart describing the experimental design in the DSS-induced colitis mouse model. DSS was dissolved in drinking water and fed on the 1^st and 4^th week. On the 2^nd, 3^rd, 5^th, and 6^th week, mice in the experimental groups were orally administrated with PBS (NS), RSV, Li01, or RSV+Li01. (b–d) Body weight changes, spleen weight, and colon length were recorded and analyzed. (e) H&E staining and colonic damage scores for colon tissue of different groups. Scale bars: 100 μm. (f) Expression of claudin, occluding, and ZO-1 in colon tissue evaluated by immunofluorescence. Scale bars, 100 μm. (g) Concentration of proinflammatory and anti-inflammatory cytokines were analyzed in the serum. Data are presented as mean ± SEM, n = 5, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 for the comparison.

Figure 2

Alterations of gut microbiota and metabolic profiles in stool samples after different treatments in DSS-induced colitis mice. (a) β-Diversity of the gut microbiota by PCoA plots. (b, c) Relative abundance of microbial taxa at the genus level in each group. (d) Concentrations of SCFAs were measured (n = 5). (e) Analysis of various metabolites in metabolic pathway between NS and RSV+Li01 groups and the difference of tryptophan and indoleacetic acid concentration in all groups. Data are presented as mean ± SEM, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 for the comparison.

Figure 3

Interactions between RSV and Li01 through in vivo and in vitro studies. (a) Impact of RSV on the growth curve of Li01. (b) Experimental design for investigating the change in gut microbiota at the genus level of conventional mice subjected to different treatments, including RSV, Li01, and RSV+Li01. (c) Experimental design for studying the metabolism of RSV in GF mice before and after colonization with Li01. The Li01 colonies in feces collected on days 4, 7, and 14 in GF mice were shown. (d) Concentration of RSV and its metabolites were measured in feces of GF, conventional mice, and DSS-induced colitis mice (n = 5). Data are presented as mean ± SEM, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 for the comparison.

Figure 4

RSV and Li01 combination treatment improved intestinal barrier function by activating the expression of AHR. Relative mRNA levels of AHR (a) and CYP1A1 (b) in colon tissue of GF mice, conventional mice, and DSS-induced colitis mice were determined by RT-qPCR. Effect of RSV and DHR on the expression of AHR and CYP1A1 in Caco-2 monolayer cells by RT-qPCR analysis (c) and Western blot analysis (d). Data are presented as mean ± SEM, n = 5, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 for the comparison.

Figure 5

Impact of RSV and Li01 combination treatment on the serotonergic pathway in DDS-induced colitis mice. (a) 5-HT concentration in serum was quantified. The expression of Tph1, SERT, and 5-HT₇R in the colon of DSS-induced colitis mice were analyzed by RT-qPCR. The expression of Tph1, SERT, and 5-HT₇R in Caco-2 monolayer cells were determined by RT-qPCR analysis (b) and Western blot analysis (c). Data are presented as mean ± SEM, n = 5, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 for the comparison.

Figure 6

A schematic diagram illustrating the mechanism of synergistic effects of RSV and Li01 combination in treatment ameliorating DSS-induced colitis.