Resveratrol alleviates DSS-induced IBD in mice by regulating the intestinal microbiota-macrophage-arginine metabolism axis

Abstract

Background: Inflammatory bowel disease (IBD) is a global disease with a growing public health concern and is associated with a complex interplay of factors, including the microbiota and immune system. Resveratrol, a natural anti-inflammatory and antioxidant agent, is known to relieve IBD but the mechanism involved is largely unexplored.

Methods: This study examines the modulatory effect of resveratrol on intestinal immunity, microbiota, metabolites, and related functions and pathways in the BALB/c mice model of IBD. Mouse RAW264.7 macrophage cell line was used to further explore the involvement of the macrophage-arginine metabolism axis. The treatment outcome was assessed through qRT-PCR, western blot, immunofluorescence, immunohistochemistry, and fecal 16S rDNA sequencing and UHPLC/Q-TOF-MS.

Results: Results showed that resveratrol treatment significantly reduced disease activity index (DAI), retained mice weight, repaired colon and spleen tissues, upregulated IL-10 and the tight junction proteins Occludin and Claudin 1, and decreased pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Resveratrol reduced the number of dysregulated metabolites and improved the gut microbial community structure and diversity, including reversing changes in the phyla Bacteroidetes, Proteobacteria, and Firmicutes, increasing 'beneficial' genera, and decreasing potential pathogens such as Lachnoclostridium, Acinobacter, and Serratia. Arginine-proline metabolism was significantly different between the colitis-treated and untreated groups. In the colon mucosa and RAW264.7 macrophage, resveratrol regulated arginine metabolism towards colon protection by increasing Arg1 and Slc6a8 and decreasing iNOS.

Conclusion: This uncovers a previously unknown mechanism of resveratrol treatment in IBD and provides the microbiota-macrophage-arginine metabolism axis as a potential therapeutic target for intestinal inflammation.

Keywords: Arginine metabolism; Inflammatory bowel disease; Macrophage; Microbiota; Resveratrol.

Fig. 1

Resveratrol relieves the features of colitis in mice. A Schematic diagram of animal model construction. B Percentage of weight loss in each group during modeling. C Disease Activity Index score. D: General view of the colorectum. E General view of spleen. F Western blot detection of PCNA、Occludin、Claudin1 expression in colon tissues. G HE staining of colon tissue (200 ×). H HE staining of spleen tissue (100 ×). I qRT-PCR detection of cytokines expression in colon tissues. * represents p < 0.05, ** represents p < 0.01 and *** represents p < 0.001

Fig. 2

Resveratrol improves gut microbial general community structure and diversity. A Venn diagram of OTUs between the DSS group, the RSV group, and the control. B Community abundance of the top 10 phyla within the groups. C Cluster heatmap of species abundance at the family level within the groups. D Cluster heatmap of species abundance at the phyla level within the groups. E Goods coverage box chart of the difference between α diversity index of the groups. F Group difference analysis of β diversity based on Unweighted Unifrac distance

Fig. 3

Resveratrol modulates specific bacteria species and restores functional dysregulation. A Cladogram (evolutionary branch diagram) of statistically different microbiota within each group. B–E Comparison of abundances of bacterial markers with significant differences between NC, DSS, and RSV groups (Provotella_9,Lachnoclostridium, Ruminococcaceae_UCG_005, and Serratia). F STAMP t-test of species with significant differences at the genus level between the DSS and NC groups. G KEGG heatmap prediction of the function of differential bacteria between the RSV and DSS group. H STAMP t-test of species with significant differences at the genus level between the RSV and NC groups. I KEGG LDA score of predicted functions of the DSS vs NC group

Fig. 4

Resveratrol reduces gut metabolites dysregulation. A Venn diagram of differential metabolites within different groups in negative ion mode. B Venn diagram of differential metabolites within different groups in positive ion mode. C PCA score map in negative ion mode. D PCA score map in positive ion mode. E Volcano plot of differential metabolites between the DSS and NC groups in positive ion mode. F Volcano plot of differential metabolites between the RSV and NC groups in positive ion mode. G Multiple analysis of significant differences in metabolite expression in positive ion mode between the DSS and NC groups. H Multiple analysis of significant differences in metabolite expression in positive ion mode between the RSV and NC groups

Fig. 5

Resveratrol modulates the arginine/proline metabolic pathway. A Hierarchical clustering heatmap of significantly differential metabolites in negative ion mode. B Hierarchical clustering heatmap of significantly differential metabolites in positive ion mode. C Differential metabolite network diagram between the RSV group and the DSS group in positive ion mode. D KEGG enrichment pathway diagram (bubble diagram). E Differential abundance score plot of all enriched metabolic pathways between the RSV group and the DSS group. F Clustering heatmap of differential metabolites in the KEGG pathway for arginine and proline metabolism between the DSS and the RSV groups. G KEGG pathway diagram of arginine and proline metabolism containing differential metabolites

Fig. 6

Resveratrol modulates the microbiota-arginine/proline metabolic axis. A Spearman correlation analysis of hierarchical clustering heatmap of significant difference microbiota and significant difference metabolites betweenthe RSV and DSS groups. P-value reflects the significant level of correlation and was defined by P < 0.05 as *, P < 0.01 as **, P < 0.001 as ***. B Spearman correlation analysis network of significant difference flora and metabolites between the RSV and DSS groups. In the correlation network diagram, the color of the line represents the positive and negative value of the correlation coefficient between the two (blue represents negative correlation and red represents positive correlation), and the thickness of the line is directly proportional to the absolute value of the correlation coefficient. The node size is positively correlated with its degree, that is, the greater the degree, the larger the node size; C–F Representative scatter diagram of correlation (Creatine, Sarcosine,4-acetamidobutanoate, Creatinine)

Fig. 7

Resveratrol regulates arginine metabolism in mice colon. A–C qRT-PCR analysis of the mRNA expression level of the arginine metabolism-related molecules (iNOS, Arg1, Slc6a8) in mouse colon tissues. D–E Western blot analysis of the protein expression level of arginine metabolism-related molecules in mouse colon tissues and its grayscale scanning analysis. F IHC analysis of Slc6a8 expression in mouse colon tissues (200 ×). G Representative images of IF staining for iNOS and Arg1 on sections of mouse colon tissues (200 ×). * represents p < 0.05, ** represents p < 0.01 and *** represents p < 0.001

Fig. 8

Resveratrol regulates arginine metabolism in macrophages in vitro. A–D qRT-PCR detection of cytokines expression in RAW264.7 cells. E–G qRT-PCR analysis of the mRNA expression level of the mRNA level of arginine metabolism-related molecules (iNOS, Arg1, Slc6a8) in RAW264.7 cells. H, I Western blot analysis of the protein expression level of arginine metabolism-related molecules in RAW264.7 cells and its grayscale scanning analysis. * represents p < 0.05, ** represents p < 0.01 and *** represents p < 0.001

Fig. 9

Summary of the mechanism of resveratrol-mediated colitis