Sedanolide alleviates DSS-induced colitis by modulating the intestinal FXR-SMPD3 pathway in mice

Abstract

Introduction: Inflammatory bowel disease (IBD) is a global disease with limited therapy. It is reported that sedanolide exerts anti-oxidative and anti-inflammatory effects as a natural phthalide, but its effects on IBD remain unclear.

Objectives: In this study, we investigated the impacts of sedanolide on dextran sodium sulfate (DSS)-induced colitis in mice.

Methods: The mice were administered sedanolide or vehicle followed by DSS administration, after which colitis symptoms, inflammation levels, and intestinal barrier function were evaluated. Transcriptome analysis, 16S rRNA sequencing, and targeted metabolomics analysis of bile acids and lipids were performed.

Results: Sedanolide protected mice from DSS-induced colitis, suppressed the inflammation, restored the weakened epithelial barrier, and modified the gut microbiota by decreasing bile salt hydrolase (BSH)-expressing bacteria. The downregulation of BSH activity by sedanolide increased the ratio of conjugated/unconjugated bile acids (BAs), thereby inhibiting the intestinal farnesoid X receptor (FXR) pathway. The roles of the FXR pathway and gut microbiota were verified using an intestinal FXR-specific agonist (fexaramine) and germ-free mice, respectively. Furthermore, we identified the key effector ceramide, which is regulated by sphingomyelin phosphodiesterase 3 (SMPD3). The protective effects of ceramide (d18:1/16:0) against inflammation and the gut barrier were demonstrated in vitro using the human cell line Caco-2.

Conclusion: Sedanolide could reshape the intestinal flora and influence BA composition, thus inhibiting the FXR-SMPD3 pathway to stimulate the synthesis of ceramide, which ultimately alleviated DSS-induced colitis in mice. Overall, our research revealed the protective effects of sedanolide against DSS-induced colitis in mice, which indicated that sedanolide may be a clinical treatment for colitis. Additionally, the key lipid ceramide (d18:1/16:0) was shown to mediate the protective effects of sedanolide, providing new insight into the associations between colitis and lipid metabolites.

Keywords: Bile acid metabolism; Ceramide; Colitis; FXR; Gut microbes; Sedanolide.

Graphical abstract

Fig. 1

Sedanolide alleviated DSS-induced colitis in mice. (A) Experimental scheme. (B) Percent of initial weight (left) and DAI score (right) of mice after DSS administration. (C) Representative photos of colon (left) and colon length (right) of mice at the last day. (D) Representative images of H&E staining of colon tissues (scale bar: 100 μm). (E) Relative expression of IL-1β, IL-6 and TNF-α in colon tissues. (F) The level of FITC-dextran in serum. (G) Relative expression of ZO-1 and MUC2) in colon tissues. (H) Representative images of immunofluorescence staining for Occludin and ZO-1 in colon tissues (scale bar: 50 μm). Red indicates positive cells. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Sedanolide modulated multiple metabolic pathways in DSS-treated mice, especially the bile acid metabolism. (A) Principal component analysis (PCA) plot of transcriptome. (B-D) Enriched signaling pathway using GSEA. (E) Top 20 enriched pathway based on KEGG analysis. (F) Heatmap of FXR-related genes.

Fig. 3

Sedanolide inhibited the BSH activity and regulated bile acid metabolism. (A) Composition of bile acids (Top 10) in colonic contents. (B) Total bile acids in colonic contents. (C) Total primary bile acids (left) and total secondary bile acids (right) in colonic contents. (D) The ratio of primary/secondary bile acids (left) and the ratio of conjugated/unconjugated bile acids. (E) BSH activity of colonic contents. (F) Concentrations of bile acids profiles. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Activated FXR pathway counteracted the protective effects of sedanolide in DSS-induced colitis mice. (A) Relative expression of FXR-related genes in colon tissues (FXR, FGF15 and SHP). (B) Experimental scheme. (C) Percent of initial weight of mice after DSS administration. (D) DAI score of mice after DSS administration. (E) Colon length (left) and representative photos of colon (right) of mice at the last day. (F) Representative H&E staining of colon tissues (scale bar: 100 μm). (G) The level of FITC-dextran in serum. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Sedanolide reshaped the BAs metabolism-related gut microbes. (A) alpha diversity indexes (Observed species, Shannon and chao1) of mice. (B) Principal coordinates analysis (PCoA) plot of microbiota. (C) Relative abundance of bacteria (Top 15) at the family level. (D) Relative abundance of bacteria (Top 15) at the genus level. (E) Relative abundance of specific bacteria. (F) Relative abundance of bile acids-related bacteria. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

Gut microbiota played a vital role in the protective effect of sedanolide against DSS-induced colitis in mice. (A) Experimental scheme. (B) Percent of initial weight of mice after DSS administration (left) and the weight of mice at the last day (right). (C) DAI score of mice after DSS administration. (D) The level of IL-6 and TNF-α in the serum. (E) Colon length of mice at the last day. (F) The level of FITC-dextran in serum. (G) Representative images of H&E staining of colon tissues (scale bar: 100 μm) and representative images of immunohistochemical staining for MPO, Occludin and ZO-1 in colon tissues (scale bar: 50 μm). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Fig. 7

Ceramide (d18:1/16:0) mediated the protective effects of sedanolide as the key effector of the FXR-SMPD3 pathway. (A) Relative expression of SMPD3 in colon tissues. (B) The pathway enrichment analysis based on differential lipids (Top 20). (C) Principal component analysis (PCA) plot of ceramides. (D) Relative abundance of total ceramides in colon. (E) Relative abundance of ceramides profiles in colon. (F) The VIP value of specific ceramides (Top 10). (G) Relative abundance of ceramide (d18:1/16:0). (H) The level of IL-1β in Caco-2 cells with different concentrations of LPS for 24 h exposure. (I) Cell viability of Caco-2 cells after different treatments for 24 h exposure. (J) Relative expression of IL-1β, occludin and ZO-1 in Caco-2 cells. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.