Sedanolide alleviates DSS-induced colitis by modulating the intestinal FXR-SMPD3 pathway in mice
Inflammatory bowel disease (IBD) is a disease spectrum characterized by chronic and relapsing inflammation of the intestine and primarily includes Crohn’s disease (CD) and ulcerative colitis (UC) , . Nowadays, the prevalence rate of IBD remains a high level in developed countries and is rapidly increasing in developing countries, suggesting that IBD is a global disease , . However, the underlying etiology of IBD, which involves interactions among genetic background, environment, lifestyle, microbiome, and immune responses, has not been determined . Traditional treatments for IBD, such as 5-aminosalicylic acid (5-ASA), are effective for treating IBD, and while current nonbiological drugs alleviate clinical symptoms, they cannot fundamentally change the course of IBD. Biological treatments such as monoclonal antibodies against cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-12, IL-23) are effective in the induction and maintenance phases of IBD and even hold promise for influencing the course of the disease and improving the quality of life of IBD patients , . However, many patients are primary or secondary non-responders to anti-TNF-α therapy in clinical trials , , , and the adverse effects associated with infections and malignancies cannot be ignored , . As the pathogenesis of IBD has been increasingly recognized by researchers, a number of novel therapeutic approaches, such as fecal microbiota transplantation (FMT), probiotics, diet administration and stem cells, have emerged. However, these novel treatments still require investigation and further verification in clinical trials , , . As a result, the clinical management of IBD remains limited. Due to the high incidence of IBD and inadequate treatments available, novel and efficient therapies for IBD are urgently needed. To evaluate the effects of various potential therapies, several models of colitis have been established. The dextran sulfate sodium (DSS)-induced colitis model has been widely used for its similarity to human IBD . Many potential treatments for IBD have been examined using the DSS-induced colitis model. Recently, researchers have shown that natural compounds such as Huangqi decoction, modified Gegen Qinlian decoction, rutaecarpine and mulberry anthocyanins ameliorate DSS-induced colitis by restoring the intestinal epithelial barrier and modulating related signaling pathways , , , . Monosaccharides such as mannose and polysaccharides such as Dendrobium fimbriatum hook polysaccharide were shown to ameliorate DSS-induced intestinal mucosal injury. In addition, the gut microbiota plays a vital role in the protective effects of potential drugs such as berberine and green tea polyphenols against DSS-induced colitis. Human umbilical cord mesenchymal stem cells were shown to protect against DSS-induced colitis , and proteins, such as secreted glucose-regulated protein 78 and mesencephalic astrocyte-derived neurotrophic factor, were reported to protect mice against the DSS-induced colitis , . Related genes have also been identified. It was reported that Gpr174 knockout and METTL3 knockdown alleviated DSS-induced colitis, while MCPIP1 and Ring1a restrained mucosal inflammation. Currently, researchers have demonstrated that inflammatory responses in the gut could result in reactive oxygen species (ROS) production and trigger oxidative damage , which highlights a new research direction. Natural products are widely used in the food industry because of their low toxicity and multiple functions . Sedanolide, which is a naturally occurring phthalide-like compound derived from celery seed oil , , has been reported to exert antioxidant effects, including increasing glutathione S-transferase (GST) activity and inhibiting tumor incidence . In addition, previous studies have indicated that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway to protect mice from acetaminophen-induced liver injury and is also associated with bacteria such as Lactobacillus . Prebiotics such as polysaccharides, can interact with the gut microbiota and treat intestinal diseases such as colitis . Based upon these findings, we hypothesized that sedanolide could ameliorate oxidative stress and inflammatory diseases, through a mechanism dependent on the gut microbiota. However, its effect on IBD is still unclear. In this study, we investigated the impacts of sedanolide on DSS-induced colitis in mice. A reduction in inflammation and strengthening of the intestinal barrier were observed in sedanolide-treated mice with colitis. Moreover, sedanolide reshaped the intestinal microbiome and bile acids (BAs) and influenced the intestinal FXR-SMPD3 pathway. Finally, ceramide (d18:1/16:0) was demonstrated to alleviate colitis as a key effector of sedanolide. 

Detailed information about the materials and instruments used in our study is listed in and , respectively. Mice To investigate the effects of sedanolide on mice with DSS-induced colitis, 6-week-old male C57BL/6J mice (GemPharmatech, Nanjing, China) were maintained in an SPF environment with adequate water and food. The mice were exposed to a 12 h light/12 h dark cycle and a temperature of 23 ± 2 °C and were randomly divided into four groups: the NC, PC, Se5, and Se20 groups. The mice were treated with sedanolide (5 or 20 mg/kg) or vehicle daily by oral gavage for 14 days. Sedanolide (GLPBIO, California, USA) was dissolved in a vehicle composed of 1 % dimethyl sulfoxide (DMSO) and 99 % phosphate buffered saline (PBS). 2 % DSS (MP Biomedicals, California, USA) was used to induce colitis as previously reported . From Day 7 to Day 14, mice in the PC, Se5, and Se20 groups were administered 2 % DSS. On Day 15, the mice were administered water instead of 2 % DSS and subsequently euthanized with anesthesia on Day 16. Blood was collected and centrifuged at 3,000 rpm for 15 min at 4 °C. Then, the serum was harvested and stored at −80 °C until analysis. The contents of the colon and cecum, as well as the colon tissue, were collected and stored at −80 °C until further use. To examine the role of the FXR pathway in the protective effects of sedanolide against DSS-induced colitis, 6-week-old male C57BL/6J mice were randomly divided into four groups: the D, FD, SD, and SFD groups. Each group was administered sedanolide (20 mg/kg), fexaramine (100 mg/kg), their combination or the vehicle by gavage daily for 14 days . Fexaramine (Biochempartner, Shanghai, China) was dissolved in a vehicle consisting of 0.5 % sodium carboxymethyl cellulose (CMC) and 99.5 % PBS. From Day 7 to Day 14, the mice in the groups were administered 2 % DSS solution. Subsequently, the mice were sacrificed as described previously. Germ-free (GF) mice are the model system to investigate the effect of specific gut microbe on host physiology . To evaluate the role of gut microbes in the protective effects of sedanolide on mice with DSS-induced colitis, male ICR GF mice aged 6–7 weeks were randomly divided into three groups: the GF, GD, and GSD groups. Establishment and maintenance of GF mice were performed as previously reported . The mice were administered sedanolide (20 mg/kg) or vehicle by gavage for 14 days. The mice in the GD and GSD groups were administered 2 % DSS from Day 7 to Day 14 to induce colitis. Then, the mice were sacrificed as described previously. Ethics statement All experiments involving animals were conducted according to ethical policies and procedures approved by the Animal Care and Use Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (Approval no. 2023–993). Cell experiments In vitro, lipopolysaccharides (LPS)-stimulated human colorectal adenocarcinoma cell line Caco-2 was used as an inflammatory colon cell model according to a previous study . The Caco-2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, MD, USA) supplemented with 15 % fetal bovine serum (FBS, Opcel Biotechnology Co.,Ltd, Inner Mongolia, China) and 1 % penicillin/streptomycin (Gibco) in an incubator with 5 % CO 2 at 37 °C as previously reported . To examine the tolerance of Caco-2 cells to LPS-induced inflammation, Caco-2 cells were seeded into 12-well plates for 24 h, and then treated with 0, 0.1, 1, 10 or 100 (μg/ mL) LPS for 24 h. The concentration range of LPS to induce cellular inflammation was referenced from previous study . Then, a Human IL-6 ELISA Kit (Abcam, Cambridge, UK) was used to measure cytokine levels according to the manufacturer’s instructions. To examine the effects of ceramide (d18:1/16:0) on lipopolysaccharide (LPS)-induced cellular injury, Caco-2 cells were seeded into 96-well plates. After adhering, the cells were exposed to 1 μg/mL LPS for 24 h, meanwhile, 10, 50, 100, 500 (ng/mL), or 1 μg/mL ceramide (d18:1/16:0) was added to the cultures. A Cell Counting Kit-8 (Beyotime, Shanghai, China) was used to examine cell viability according to the manufacturer’s instructions. To investigate the effects of ceramide (d18:1/16:0) on LPS-induced cellular inflammation, Caco-2 cells were seeded into 6-well plates and divided into four groups: the control, Cer 16:0 (50 ng/mL ceramide [d18:1/16:0]), LPS (1 μg/mL LPS) and LPS + Cer 16:0 (1 μg/mL LPS + 50 ng/mL ceramide [d18:1/16:0] groups. After 24 h of incubation, the cells were harvested for real-time polymerase chain reaction (PCR) analysis. Assessment of colitis severity According to the disease activity index (DAI) scoring system, mouse bodyweight and the characteristics of their feces, especially fecal blood and consistency, were recorded from Day 8 to Day 15. These three indicators can reflect the severity of DSS-induced colitis, and each were assigned a score from 0 to 4, the sum of which resulting in the DAI score . The occult blood test was carried out using a fecal occult blood reagent (Baso, Zhuhai, China). On the final day, colon length was measured after the mice were sacrificed. Histological examination, immunofluorescence analysis and immunohistochemical staining The distal colon segment was fixed in 10 % neutral buffered formalin for 24 h, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and finally scanned with a microscope. Immunofluorescence staining and immunohistochemical staining for Zona Occludens 1 (ZO-1) and occludin were performed using the indicated antibodies (Proteintech, Chicago, USA). Immunohistochemical staining for myeloperoxidase (MPO) was performed using an anti-MPO antibody (Proteintech, Chicago, USA) according to the standard procedure. Fluorescein isothiocyanate (FITC)-dextran permeability assay FITC-dextran (Sigma-Aldrich, St. Louis, USA) permeability was assessed as previously reported .On Day 15, mice were administered 44 mg FITC-dextran /100 g body weight by gavage after an overnight fast and 4 h later, serum was collected in the dark. A fluorescence microplate reader (485/528 nm) was used to measure the concentration of FITC. Serum cytokines analysis Serum levels of IL-6 and TNF-α were assessed by an IL-6 ELISA Kit and a TNF-α ELISA Kit (MLbio, Shanghai, China) according to the manufacturer’s instructions. Bile salt hydrolase (bsh) activity analysis Based on previous studies , , , BSH activity was assessed by measuring the release of taurine through the deconjugation of conjugated bile acids (BAs). 50 mg of colonic contents was homogenized with 250 mL of radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) and centrifuged at 15,000 rpm and 4 °C for 15 min to extract total proteins, after which a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China) was used to quantify the extracted proteins. The proteins were subsequently diluted to 1 mg/mL with 3 mM sodium acetate solution. Then, 3 mM sodium acetate (170 μL) and 1 mM taurodeoxycholic acid (TDCA) or tauroursodeoxycholic acid (TUDCA) (20 μL) were added to the protein extracts (10 μL), which were subsequently incubated at 37 °C for 30 min. The reaction was stopped by placing the samples in dry ice. The thawed mixture was centrifuged, and the supernatant (20 μL) was collected. Additional experimental details can be found in a previous study . Real-time PCR Ribonucleic acid (RNA) was extracted from colon tissue or cells with an RNeasy Plus Mini Kit (Qiagen, CA, USA). The extracted RNA was then reverse transcribed to complementary deoxyribonucleic acid (cDNA) using a PrimeScript™ RT reagent kit (Takara, Kusatsu, Japan). Then, SYBR Premix Ex Taq II reagent (Takara) was used to measure the relative expression of the target genes with a ViiA7 real-time PCR system (Applied Biosystems) based on the 2 −ΔΔCT method (GAPDH was used as the control). The sequences of the primers used are listed in . Statistical analysis The data are presented as the mean ± SEM. GraphPad Prism v.8 (GraphPad Software Inc., San Diego, USA) was used for the statistical analysis. To test the differences, Student’s t test was used to compare two groups with normal distributions; otherwise, the Mann-Whitney U test was used. Ordinary analysis of variance (ANOVA) was used for comparisons among more than two groups with equal standard deviations; otherwise, Brown-Forsythe and Welch ANOVA were performed. A P value < 0.05 was considered to indicate statistical significance. 

To investigate the effects of sedanolide on mice with DSS-induced colitis, 6-week-old male C57BL/6J mice (GemPharmatech, Nanjing, China) were maintained in an SPF environment with adequate water and food. The mice were exposed to a 12 h light/12 h dark cycle and a temperature of 23 ± 2 °C and were randomly divided into four groups: the NC, PC, Se5, and Se20 groups. The mice were treated with sedanolide (5 or 20 mg/kg) or vehicle daily by oral gavage for 14 days. Sedanolide (GLPBIO, California, USA) was dissolved in a vehicle composed of 1 % dimethyl sulfoxide (DMSO) and 99 % phosphate buffered saline (PBS). 2 % DSS (MP Biomedicals, California, USA) was used to induce colitis as previously reported . From Day 7 to Day 14, mice in the PC, Se5, and Se20 groups were administered 2 % DSS. On Day 15, the mice were administered water instead of 2 % DSS and subsequently euthanized with anesthesia on Day 16. Blood was collected and centrifuged at 3,000 rpm for 15 min at 4 °C. Then, the serum was harvested and stored at −80 °C until analysis. The contents of the colon and cecum, as well as the colon tissue, were collected and stored at −80 °C until further use. To examine the role of the FXR pathway in the protective effects of sedanolide against DSS-induced colitis, 6-week-old male C57BL/6J mice were randomly divided into four groups: the D, FD, SD, and SFD groups. Each group was administered sedanolide (20 mg/kg), fexaramine (100 mg/kg), their combination or the vehicle by gavage daily for 14 days . Fexaramine (Biochempartner, Shanghai, China) was dissolved in a vehicle consisting of 0.5 % sodium carboxymethyl cellulose (CMC) and 99.5 % PBS. From Day 7 to Day 14, the mice in the groups were administered 2 % DSS solution. Subsequently, the mice were sacrificed as described previously. Germ-free (GF) mice are the model system to investigate the effect of specific gut microbe on host physiology . To evaluate the role of gut microbes in the protective effects of sedanolide on mice with DSS-induced colitis, male ICR GF mice aged 6–7 weeks were randomly divided into three groups: the GF, GD, and GSD groups. Establishment and maintenance of GF mice were performed as previously reported . The mice were administered sedanolide (20 mg/kg) or vehicle by gavage for 14 days. The mice in the GD and GSD groups were administered 2 % DSS from Day 7 to Day 14 to induce colitis. Then, the mice were sacrificed as described previously. 

All experiments involving animals were conducted according to ethical policies and procedures approved by the Animal Care and Use Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (Approval no. 2023–993). 

In vitro, lipopolysaccharides (LPS)-stimulated human colorectal adenocarcinoma cell line Caco-2 was used as an inflammatory colon cell model according to a previous study . The Caco-2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, MD, USA) supplemented with 15 % fetal bovine serum (FBS, Opcel Biotechnology Co.,Ltd, Inner Mongolia, China) and 1 % penicillin/streptomycin (Gibco) in an incubator with 5 % CO 2 at 37 °C as previously reported . To examine the tolerance of Caco-2 cells to LPS-induced inflammation, Caco-2 cells were seeded into 12-well plates for 24 h, and then treated with 0, 0.1, 1, 10 or 100 (μg/ mL) LPS for 24 h. The concentration range of LPS to induce cellular inflammation was referenced from previous study . Then, a Human IL-6 ELISA Kit (Abcam, Cambridge, UK) was used to measure cytokine levels according to the manufacturer’s instructions. To examine the effects of ceramide (d18:1/16:0) on lipopolysaccharide (LPS)-induced cellular injury, Caco-2 cells were seeded into 96-well plates. After adhering, the cells were exposed to 1 μg/mL LPS for 24 h, meanwhile, 10, 50, 100, 500 (ng/mL), or 1 μg/mL ceramide (d18:1/16:0) was added to the cultures. A Cell Counting Kit-8 (Beyotime, Shanghai, China) was used to examine cell viability according to the manufacturer’s instructions. To investigate the effects of ceramide (d18:1/16:0) on LPS-induced cellular inflammation, Caco-2 cells were seeded into 6-well plates and divided into four groups: the control, Cer 16:0 (50 ng/mL ceramide [d18:1/16:0]), LPS (1 μg/mL LPS) and LPS + Cer 16:0 (1 μg/mL LPS + 50 ng/mL ceramide [d18:1/16:0] groups. After 24 h of incubation, the cells were harvested for real-time polymerase chain reaction (PCR) analysis. 

According to the disease activity index (DAI) scoring system, mouse bodyweight and the characteristics of their feces, especially fecal blood and consistency, were recorded from Day 8 to Day 15. These three indicators can reflect the severity of DSS-induced colitis, and each were assigned a score from 0 to 4, the sum of which resulting in the DAI score . The occult blood test was carried out using a fecal occult blood reagent (Baso, Zhuhai, China). On the final day, colon length was measured after the mice were sacrificed. 

The distal colon segment was fixed in 10 % neutral buffered formalin for 24 h, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and finally scanned with a microscope. Immunofluorescence staining and immunohistochemical staining for Zona Occludens 1 (ZO-1) and occludin were performed using the indicated antibodies (Proteintech, Chicago, USA). Immunohistochemical staining for myeloperoxidase (MPO) was performed using an anti-MPO antibody (Proteintech, Chicago, USA) according to the standard procedure. 

FITC-dextran (Sigma-Aldrich, St. Louis, USA) permeability was assessed as previously reported .On Day 15, mice were administered 44 mg FITC-dextran /100 g body weight by gavage after an overnight fast and 4 h later, serum was collected in the dark. A fluorescence microplate reader (485/528 nm) was used to measure the concentration of FITC. 

Serum levels of IL-6 and TNF-α were assessed by an IL-6 ELISA Kit and a TNF-α ELISA Kit (MLbio, Shanghai, China) according to the manufacturer’s instructions. 

Based on previous studies , , , BSH activity was assessed by measuring the release of taurine through the deconjugation of conjugated bile acids (BAs). 50 mg of colonic contents was homogenized with 250 mL of radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) and centrifuged at 15,000 rpm and 4 °C for 15 min to extract total proteins, after which a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China) was used to quantify the extracted proteins. The proteins were subsequently diluted to 1 mg/mL with 3 mM sodium acetate solution. Then, 3 mM sodium acetate (170 μL) and 1 mM taurodeoxycholic acid (TDCA) or tauroursodeoxycholic acid (TUDCA) (20 μL) were added to the protein extracts (10 μL), which were subsequently incubated at 37 °C for 30 min. The reaction was stopped by placing the samples in dry ice. The thawed mixture was centrifuged, and the supernatant (20 μL) was collected. Additional experimental details can be found in a previous study . 

Ribonucleic acid (RNA) was extracted from colon tissue or cells with an RNeasy Plus Mini Kit (Qiagen, CA, USA). The extracted RNA was then reverse transcribed to complementary deoxyribonucleic acid (cDNA) using a PrimeScript™ RT reagent kit (Takara, Kusatsu, Japan). Then, SYBR Premix Ex Taq II reagent (Takara) was used to measure the relative expression of the target genes with a ViiA7 real-time PCR system (Applied Biosystems) based on the 2 −ΔΔCT method (GAPDH was used as the control). The sequences of the primers used are listed in . 

The data are presented as the mean ± SEM. GraphPad Prism v.8 (GraphPad Software Inc., San Diego, USA) was used for the statistical analysis. To test the differences, Student’s t test was used to compare two groups with normal distributions; otherwise, the Mann-Whitney U test was used. Ordinary analysis of variance (ANOVA) was used for comparisons among more than two groups with equal standard deviations; otherwise, Brown-Forsythe and Welch ANOVA were performed. A P value < 0.05 was considered to indicate statistical significance. 

Sedanolide protected mice from DSS-induced colitis, attenuated intestinal inflammation and strengthened the gut barrier In order to evaluate the impacts of different concentrations of sedanolide on DSS-induced colitis, mice were treated with 5 or 20 mg/kg sedanolide ( A). During the experiment, DSS significantly decreased the weight of the mice and markedly increased the DAI score; however, 20 mg/kg sedanolide (SE20) significantly increased body weight and decreased the DAI score in DSS-treated mice ( B). Moreover, SE20 significantly increased the colon length, which was shortened by DSS ( C). HE staining revealed that DSS led to severe inflammation, epithelial erosion and loss, crypt damage, and goblet cell loss, and these effects could be alleviated by sedanolide in a dose-dependent manner ( D). Mice in the lower concentration group (SE5) experienced some protection compared to those in the SE20 group. Although noticeable changes were observed for these indicators, there were no significant differences. According to previous researches, DSS has been shown to cause colonic inflammation and weaken intestinal barrier function , . In our study, DSS significantly up-regulated IL-1β , IL-6, and TNF-α mRNA levels in colon tissue, and sedanolide significantly reduced colonic inflammation ( E). On the other hand, DSS increased intestinal permeability, as indicated by the increased serum levels of FITC-dextran ( F) and decreased colonic mRNA levels of ZO-1 ( G). However, sedanolide restored intestinal permeability and strengthened the intestinal barrier in DSS-treated mice. In addition, serum levels of FITC-dextran in the SE20 group were markedly lower than those in the PC group, and the changes in the mRNA expression of ZO-1 and MUC2 mRNAs were the same, as shown in F-1G. Colonic immunofluorescence staining for ZO-1 and Occludin demonstrated consistent changes ( H). Transcriptome analysis revealed that multiple metabolic pathways, especially bile acid metabolism, were regulated by sedanolide To explore the impacts of sedanolide on colitis at the transcriptional level, transcriptome analysis was performed. As shown in A, distinct clustering was observed among the NC, PC and SE20 groups. The GSEA results indicated that sedanolide significantly suppressed the Janus tyrosine kinase (JAK)- signal transducer and activator of transcription (STAT) signaling pathway in the presence of DSS ( B), as well as the nuclear factor-kappa B (NF-κB) signaling pathway and genes associated with inflammatory bowel disease ( C). Moreover, notable differences were observed in cholesterol metabolism, steroid biosynthesis and taurine and hypotaurine metabolism, all of which are linked to bile acid metabolism ( D–2E). Therefore, we hypothesized that sedanolide could influence the metabolism of bile acids and performed enrichment analysis of the genes associated with bile acid metabolism ( F). As predicted, the relative intensities of FXR , FGF15 , ASBT , OSTα , IBABP and SPTLC2 in DSS-treated mice were decreased by sedanolide. Downstream genes of the FXR pathway ( SMPD3 and SMPD4 ) were upregulated by sedanolide. Sedanolide inhibited BSH activity and regulated bile acid metabolism Based on these findings, targeted quantification of bile acids (BAs) in colonic contents was performed. The top 10 BAs are shown in A. The administration of DSS markedly decreased total BA concentrations, and this effect was reversed by sedanolide ( B); and the same changes were observed in total primary BA concentrations levels and total secondary BA concentrations ( C). In addition, there was a decrease in the ratio of primary BAs to secondary BAs after DSS treatment, and sedanolide increased this ratio ( D). DSS significantly reduced the ratio of conjugated BAs to unconjugated BAs, and sedanolide resulted in an increase, as shown in D. Consistently, the ability of BSH to deconjugate TDCA and TUDCA was enhanced by DSS but was reduced by sedanolide ( E). Statistical analysis revealed significant decreases in the levels of CDCA, α-MCA, β-MCA, LCA, UDCA, TCDCA, TCA, T-α-MCA, T-β-MCA, TUDCA, TDCA and THDCA in response to DSS treatment, while the levels of α-MCA, β-MCA, LCA, UDCA, TCDCA and TDCA were markedly increased by sedanolide ( F). In conclusion, sedanolide affects bile acid metabolism, subsequently inhibiting downstream FXR genes that are activated by DSS. FXR pathway activation could counteract the protective effects of sedanolide on mice with DSS-induced colitis We further verified the expression of FXR related genes by qPCR, and found that they were upregulated by DSS and downregulated by 20 mg/kg sedanolide ( A). Then, a specific agonist of the intestinal FXR pathway (fexaramine) was used to examine the role of the colonic FXR pathway in the protective effects of sedanolide mice with DSS-induced colitis ( B). We found that the administration of fexaramine decreased body weight and increased the DAI score, which opposed the effects of sedanolide ( C–4D). In addition, the combination of sedanolide and fexaramine significantly decreased colon length in mice with colitis compared to mice that received sedanolide alone ( E). H&E staining showed that the protective effects of sedanolide were counteracted by the administration of fexaramine, resulting in more severe inflammation, more epithelial erosion and loss, as well as more crypt damage and goblet cell loss ( F). Consistently, intestinal permeability in sedanolide-treated mice was worsened by fexaramine ( G). Thus, we concluded that sedanolide protected mice from DSS-induced colitis by inhibiting of the FXR signaling pathway. Sedanolide reshaped BA metabolism-related gut microbes There is crosstalk between BAs and the gut microbiota ; therefore, we performed 16S rRNA sequencing and discovered that DSS administration markedly decreased α-diversity as shown by the Shannon index and Chao1 index. Principal coordinate analysis (PCoA) revealed that sedanolide-treated mice were distinctly clustered relative to mice with DSS-induced colitis. The effects of DSS and sedanolide on the gut microbiome at the family and the genus levels are shown in C and 5D, respectively. We selected a portion of the main flora ( E). At the phylum level, after DSS challenge, the abundance of Firmicutes and Actinobacteriota markedly decreased, and the abundance of Proteobacteria and Deferribacterota markedly increased. However, sedanolide markedly reversed the changes in the abundances of Proteobacteria , Deferribacterota and Actinobacteriota . At the family level, DSS challenge significantly decreased the abundances of Muribaculaceae , Lachnospiraceae , Prevotellaceae and Ruminococcaceae , increased the abundances of Enterobacteriaceae , Sutterellaceae and Erysipelotrichaceae. Sedanolide significantly up-regulated the abundance of Prevotellaceae , down-regulated the abundances of Enterobacteriaceae , Sutterellaceae and Erysipelotrichaceae . At the genus level, the abundances of Escherichia-Shigella and Parabacteroides were markedly increased, and the abundances of Lachnoclostridium and Alloprevotalla were markedly decreased after DSS treatment; sedanolide could partially restore the abundances of Escherichia-Shigella , Lachnoclostridium and Parabacteroides . Notably, the DSS administration increased the abundance of BSH-expressing bacteria especially Parabacteroides and Enterococcus , which could be decreased by sedanolide ( F). Moreover, sedanolide altered the levels of short-chain fatty acids (SCFAs). There were significant differences in the levels of acetic acid and butyric acid after DSS treatment, and decreases in the levels of hexanoic acid, propionic acid, and pentanoic acid were also observed. Sedanolide significantly increased the levels of acetic acid, hexanoic acid and propionic acid ( ). The gut microbiota plays a vital role in the protective effect of sedanolide against DSS-induced colitis in mice To verify the function of the gut microbiota in the protective effect of sedanolide on mice with DSS-induced colitis, GF mice were used ( A). During the experiment, DSS caused significant weight loss in GF mice with colitis, and treatment with or without sedanolide had no significant effects on weight ( B). There was also no difference in the DAI scores, or IL-6 and TNF-α levels between the GD group and GSD group ( C–6D). In GF mice, shorter colon lengths and higher serum FITC-dextran levels were observed after DSS administration, but there were no differences in colon length or serum FITC-dextran levels after sedanolide treatment ( E–6F). Histological examination and immunohistochemical staining of MPO revealed that sedanolide had a weak effect on DSS-induced intestinal disruption and inflammatory cell infiltration, and immunohistochemical staining of occludin and ZO-1 showed that sedanolide had little effect on intestinal barrier damage ( G). Thus, we concluded that the protective effect of sedanolide against DSS-induced colitis was partly mediated by the gut microbiota. Ceramide (d18:1/16:0) mediated the protective effects of sedanolide as the key effector of the FXR-SMPD3 pathway As indicated in the transcriptome analysis, SMPD3 and SMPD4, which are downstream of the FXR pathway, were activated by sedanolide. These findings indicated that sedanolide could inhibit the FXR- fibroblast growth factor 15 (FGF15) pathway and subsequently activate SMPD3 and SMPD4. RT-qPCR analysis of SMPD3 also confirmed these results ( A). SMPD3, existed in the plasma membrane, is able to use sphingomyelin to generate ceramide . Therefore, we performed targeted lipid analysis to examine the alterations in lipid profiles, especially in SMPD3-regulated ceramides. Pathway enrichment analysis based on differential lipids identified the sphingolipid signaling pathway, which also indicated a change in ceramides ( B). Obvious shifts in ceramide levels between groups were shown by principal component analysis (PCA) in C. The relative abundance of total ceramides decreased in response to DSS was increased by sedanolide ( D). By comparing the relative abundances of different classes of ceramides, we found that ceramide 16:0 was the main ceramide was significantly different between the groups ( E). Notably, we found that ceramide (d18:1/16:0) had the highest variable importance in the Projection (VIP) value among all ceramides ( F) and was also decreased by DSS and increased by sedanolide ( G). Thus, we hypothesized that ceramide(d18:1/16:0) was the key metabolite that mediated the protective effects of sedanolide. A concentration of 1 μg/mL LPS could induce significant inflammation in Caco-2 cells ( H). We treated Caco-2 cells with different concentrations of ceramide (d18:1/16:0) and 1 μg/mL LPS. As shown in I, treatment with 10 or 50 ng/mL of ceramide (d18:1/16:0) markedly increased the cell viability. Additionally, 50 ng/mL ceramide (d18:1/16:0) decreased the expression of IL-1β and increased the expression of Occludin and ZO-1 ( J), which illustrated that ceramide (d18:1/16:0) mediated the protective effects of sedanolide. 

In order to evaluate the impacts of different concentrations of sedanolide on DSS-induced colitis, mice were treated with 5 or 20 mg/kg sedanolide ( A). During the experiment, DSS significantly decreased the weight of the mice and markedly increased the DAI score; however, 20 mg/kg sedanolide (SE20) significantly increased body weight and decreased the DAI score in DSS-treated mice ( B). Moreover, SE20 significantly increased the colon length, which was shortened by DSS ( C). HE staining revealed that DSS led to severe inflammation, epithelial erosion and loss, crypt damage, and goblet cell loss, and these effects could be alleviated by sedanolide in a dose-dependent manner ( D). Mice in the lower concentration group (SE5) experienced some protection compared to those in the SE20 group. Although noticeable changes were observed for these indicators, there were no significant differences. According to previous researches, DSS has been shown to cause colonic inflammation and weaken intestinal barrier function , . In our study, DSS significantly up-regulated IL-1β , IL-6, and TNF-α mRNA levels in colon tissue, and sedanolide significantly reduced colonic inflammation ( E). On the other hand, DSS increased intestinal permeability, as indicated by the increased serum levels of FITC-dextran ( F) and decreased colonic mRNA levels of ZO-1 ( G). However, sedanolide restored intestinal permeability and strengthened the intestinal barrier in DSS-treated mice. In addition, serum levels of FITC-dextran in the SE20 group were markedly lower than those in the PC group, and the changes in the mRNA expression of ZO-1 and MUC2 mRNAs were the same, as shown in F-1G. Colonic immunofluorescence staining for ZO-1 and Occludin demonstrated consistent changes ( H). 

To explore the impacts of sedanolide on colitis at the transcriptional level, transcriptome analysis was performed. As shown in A, distinct clustering was observed among the NC, PC and SE20 groups. The GSEA results indicated that sedanolide significantly suppressed the Janus tyrosine kinase (JAK)- signal transducer and activator of transcription (STAT) signaling pathway in the presence of DSS ( B), as well as the nuclear factor-kappa B (NF-κB) signaling pathway and genes associated with inflammatory bowel disease ( C). Moreover, notable differences were observed in cholesterol metabolism, steroid biosynthesis and taurine and hypotaurine metabolism, all of which are linked to bile acid metabolism ( D–2E). Therefore, we hypothesized that sedanolide could influence the metabolism of bile acids and performed enrichment analysis of the genes associated with bile acid metabolism ( F). As predicted, the relative intensities of FXR , FGF15 , ASBT , OSTα , IBABP and SPTLC2 in DSS-treated mice were decreased by sedanolide. Downstream genes of the FXR pathway ( SMPD3 and SMPD4 ) were upregulated by sedanolide. 

Based on these findings, targeted quantification of bile acids (BAs) in colonic contents was performed. The top 10 BAs are shown in A. The administration of DSS markedly decreased total BA concentrations, and this effect was reversed by sedanolide ( B); and the same changes were observed in total primary BA concentrations levels and total secondary BA concentrations ( C). In addition, there was a decrease in the ratio of primary BAs to secondary BAs after DSS treatment, and sedanolide increased this ratio ( D). DSS significantly reduced the ratio of conjugated BAs to unconjugated BAs, and sedanolide resulted in an increase, as shown in D. Consistently, the ability of BSH to deconjugate TDCA and TUDCA was enhanced by DSS but was reduced by sedanolide ( E). Statistical analysis revealed significant decreases in the levels of CDCA, α-MCA, β-MCA, LCA, UDCA, TCDCA, TCA, T-α-MCA, T-β-MCA, TUDCA, TDCA and THDCA in response to DSS treatment, while the levels of α-MCA, β-MCA, LCA, UDCA, TCDCA and TDCA were markedly increased by sedanolide ( F). In conclusion, sedanolide affects bile acid metabolism, subsequently inhibiting downstream FXR genes that are activated by DSS. 

We further verified the expression of FXR related genes by qPCR, and found that they were upregulated by DSS and downregulated by 20 mg/kg sedanolide ( A). Then, a specific agonist of the intestinal FXR pathway (fexaramine) was used to examine the role of the colonic FXR pathway in the protective effects of sedanolide mice with DSS-induced colitis ( B). We found that the administration of fexaramine decreased body weight and increased the DAI score, which opposed the effects of sedanolide ( C–4D). In addition, the combination of sedanolide and fexaramine significantly decreased colon length in mice with colitis compared to mice that received sedanolide alone ( E). H&E staining showed that the protective effects of sedanolide were counteracted by the administration of fexaramine, resulting in more severe inflammation, more epithelial erosion and loss, as well as more crypt damage and goblet cell loss ( F). Consistently, intestinal permeability in sedanolide-treated mice was worsened by fexaramine ( G). Thus, we concluded that sedanolide protected mice from DSS-induced colitis by inhibiting of the FXR signaling pathway. 

There is crosstalk between BAs and the gut microbiota ; therefore, we performed 16S rRNA sequencing and discovered that DSS administration markedly decreased α-diversity as shown by the Shannon index and Chao1 index. Principal coordinate analysis (PCoA) revealed that sedanolide-treated mice were distinctly clustered relative to mice with DSS-induced colitis. The effects of DSS and sedanolide on the gut microbiome at the family and the genus levels are shown in C and 5D, respectively. We selected a portion of the main flora ( E). At the phylum level, after DSS challenge, the abundance of Firmicutes and Actinobacteriota markedly decreased, and the abundance of Proteobacteria and Deferribacterota markedly increased. However, sedanolide markedly reversed the changes in the abundances of Proteobacteria , Deferribacterota and Actinobacteriota . At the family level, DSS challenge significantly decreased the abundances of Muribaculaceae , Lachnospiraceae , Prevotellaceae and Ruminococcaceae , increased the abundances of Enterobacteriaceae , Sutterellaceae and Erysipelotrichaceae. Sedanolide significantly up-regulated the abundance of Prevotellaceae , down-regulated the abundances of Enterobacteriaceae , Sutterellaceae and Erysipelotrichaceae . At the genus level, the abundances of Escherichia-Shigella and Parabacteroides were markedly increased, and the abundances of Lachnoclostridium and Alloprevotalla were markedly decreased after DSS treatment; sedanolide could partially restore the abundances of Escherichia-Shigella , Lachnoclostridium and Parabacteroides . Notably, the DSS administration increased the abundance of BSH-expressing bacteria especially Parabacteroides and Enterococcus , which could be decreased by sedanolide ( F). Moreover, sedanolide altered the levels of short-chain fatty acids (SCFAs). There were significant differences in the levels of acetic acid and butyric acid after DSS treatment, and decreases in the levels of hexanoic acid, propionic acid, and pentanoic acid were also observed. Sedanolide significantly increased the levels of acetic acid, hexanoic acid and propionic acid ( ). 

To verify the function of the gut microbiota in the protective effect of sedanolide on mice with DSS-induced colitis, GF mice were used ( A). During the experiment, DSS caused significant weight loss in GF mice with colitis, and treatment with or without sedanolide had no significant effects on weight ( B). There was also no difference in the DAI scores, or IL-6 and TNF-α levels between the GD group and GSD group ( C–6D). In GF mice, shorter colon lengths and higher serum FITC-dextran levels were observed after DSS administration, but there were no differences in colon length or serum FITC-dextran levels after sedanolide treatment ( E–6F). Histological examination and immunohistochemical staining of MPO revealed that sedanolide had a weak effect on DSS-induced intestinal disruption and inflammatory cell infiltration, and immunohistochemical staining of occludin and ZO-1 showed that sedanolide had little effect on intestinal barrier damage ( G). Thus, we concluded that the protective effect of sedanolide against DSS-induced colitis was partly mediated by the gut microbiota. 

As indicated in the transcriptome analysis, SMPD3 and SMPD4, which are downstream of the FXR pathway, were activated by sedanolide. These findings indicated that sedanolide could inhibit the FXR- fibroblast growth factor 15 (FGF15) pathway and subsequently activate SMPD3 and SMPD4. RT-qPCR analysis of SMPD3 also confirmed these results ( A). SMPD3, existed in the plasma membrane, is able to use sphingomyelin to generate ceramide . Therefore, we performed targeted lipid analysis to examine the alterations in lipid profiles, especially in SMPD3-regulated ceramides. Pathway enrichment analysis based on differential lipids identified the sphingolipid signaling pathway, which also indicated a change in ceramides ( B). Obvious shifts in ceramide levels between groups were shown by principal component analysis (PCA) in C. The relative abundance of total ceramides decreased in response to DSS was increased by sedanolide ( D). By comparing the relative abundances of different classes of ceramides, we found that ceramide 16:0 was the main ceramide was significantly different between the groups ( E). Notably, we found that ceramide (d18:1/16:0) had the highest variable importance in the Projection (VIP) value among all ceramides ( F) and was also decreased by DSS and increased by sedanolide ( G). Thus, we hypothesized that ceramide(d18:1/16:0) was the key metabolite that mediated the protective effects of sedanolide. A concentration of 1 μg/mL LPS could induce significant inflammation in Caco-2 cells ( H). We treated Caco-2 cells with different concentrations of ceramide (d18:1/16:0) and 1 μg/mL LPS. As shown in I, treatment with 10 or 50 ng/mL of ceramide (d18:1/16:0) markedly increased the cell viability. Additionally, 50 ng/mL ceramide (d18:1/16:0) decreased the expression of IL-1β and increased the expression of Occludin and ZO-1 ( J), which illustrated that ceramide (d18:1/16:0) mediated the protective effects of sedanolide. 

Our research investigated the effects of the natural phthalide sedanolide on DSS-induced colitis in mice, and revealed that sedanolide could alleviate inflammation, strengthen the damaged gut barrier, reshape the imbalanced gut microbiome, influence BA metabolism and inhibit activation of the FXR pathway in DSS-treated mice. Celery seed oil is often used as an herbal medicine to treat inflammation, such as gout and rheumatism; moreover, it is a vital source of sedanolide . Consistently, we demonstrated that sedanolide played a significant role in intestinal inflammation by decreasing the expression of proinflammatory genes. Moreover, our study revealed that sedanolide possesses the ability to improve weak intestinal function by reducing excessive increase in intestinal permeability and increasing the expression of tight junction proteins and mucins in DSS-induced colitis mice. The gut microbiota has been proven to play an essential role in the pathogenesis of IBD . In previous studies, several taxa were closely linked to the progression of IBD. For example, Proteobacteria can trigger proinflammatory changes and has been consistently shown to be overrepresented in IBD patients . Additionally, the opportunistic pathogens Escherichia-Shigella and Enterobacteriaceae are linked to gut dysbiosis and induce inflammation, such as colitis , , . Our study indicated that the administration of sedanolide modulated the imbalanced gut flora in DSS-treated mice; most importantly, sedanolide decreased the abundance of opportunistic pathogenic bacteria like Proteobacteria , Escherichia-Shigella and Enterobacteriaceae . Our results are also consistent with previous studies showing that balancing Proteobacteria and Lachnospiraceae maintains gut microbe homeostasis, thus attenuating the severity of colitis . SCFAs, which are primarily acetic acid, propionic acid, and butyric acid, are produced by the fermentation of intestinal bacteria, and have been proven to reduce the symptoms and inflammation of IBD , . However, the significantly decreased taxa, including Ruminococcaceae and Lachnospiraceae, after DSS administration are SCFA-producing bacteria, and these effects could be reversed by sedanolide in our research. Mechanistically, we think that sedanolide increased the abundance of beneficial bacteria, which could competitively inhibit the proliferation and survival of harmful bacteria by competing for nutrients or secreting antimicrobial metabolites. Besides, the increase of SCFAs-producing bacteria leads to higher level of beneficial SCFAs, which is reported could strengthen the intestinal barrier . The strengthened intestinal barrier reduces the translocation of pathogenic bacteria, resulting in decreased opportunistic pathogenic bacteria. Therefore, the protective effects of sedanolide are partly mediated by maintaining the homeostasis of gut microbiome. Subsequently, we employed the germ-free mice, which are considered an appropriate model system for investigating the impacts of the intestinal flora on gastrointestinal function . Counteracting protective effects of sedanolide on DSS-treated germ-free mice verified the significant role of the intestinal microbiome in the beneficial effects of sedanolide on DSS-induced colitis mice. Based on these findings, we hypothesized that the reshaped gut microbiome could trigger the reactions in the host that make sedanolide a beneficial medicine for treating IBD. Although the reshaped gut flora is considered to be the primary driver, the molecular substances that mediate this communication have not yet been identified. Transcriptome analysis is qualified for identifying genome structure and generating biomarkers in diseases and in response to external challenges . Researchers have discovered that the JAK-STAT pathway and the NF-κB signaling pathway produce a variety of essential mediators of inflammation, including several factors involved in the progression of IBD , , . In our experiment, the JAK-STAT signaling pathway, NF-κB signaling pathway and genes associated with inflammatory bowel disease were activated by DSS while were supressed by sedanolide treatment. Notably, the administration of sedanolide influenced cholesterol metabolism, steroid biosynthesis, taurine and hypotaurine metabolism, and fat digestion and absorption. It is known to us that the synthesis and excretion of BAs are critical for cholesterol and lipid catabolism ; thus, these significantly modulated pathways are closely associated with bile acid metabolism for which it is considered a new direction for curing metabolic diseases . Besides, the production of taurine is closely linked to bile acid transformation by microbes . The transcriptome analysis suggests that BAs, as a kind of biologically active mediators, may be involved in the communication between the gut microbiome and IBD, especially in the presence of sedanolide. Previous studies have indicated that BAs are a class of steroidal molecules produced in the liver that are subsequently metabolized by gut flora-derived enzymes , and are mechanistically associated with the pathogenesis of IBD . Furthermore, the expansion of Proteobacteria and Enterobacteriaceae , the reduction in Firmicutes , including Clostridiales , and the changes in other taxa after DSS administration observed in our experiments are thought to influence the capacity of the gut flora to generate protective metabolites, especially BAs , . Taken together, these findings suggest that BAs are intermediate effectors that exert subsequent metabolism-related effects by BA-activated receptors, especially FXR , . FXR can combine with retinoid X receptor α (RXRα) to form a heterodimer that is activated by BAs in enterohepatic circulation and regulates the production of FGF15, thus performing an important role in regulating the expression of cholesterol, BAs, and lipid metabolism-related genes . Consequently, any modification of BAs, including their abundance and composition, could alter the FXR pathway and influence the progression of DSS-induced colitis. It is worth noting that bile-salt hydrolases (BSHs), which are generated by the intestinal flora, influence the transformation of conjugated BAs to deconjugated BAs as the gateway enzymes, thus playing a vital role in BA-FXR signaling . We found that the abundance of BSH-expressing bacteria, such as Parabacteroides , Enterococcus , Lactobacillus , Streptococcus and Bacillaceae, were markedly increased by DSS treatment, and were significantly decreased by sedanolide treatment in mice. Consistently, BSH activity was up-regulated by DSS and down-regulated by sedanolide, which results in more transform of conjugated BAs to deconjugated BAs in DSS-treated mice while less transform after sedanolide administration. In other words, sedanolide contributes to a higher ratio of conjugated/unconjugated BAs which is reported as an inhibiting factor of FXR . Consistent with that, our results have shown the FXR signaling pathway was activated by DSS and inhibited by sedanolide. Afterwards, a gut-restricted FXR agonist fexaramine was used in combination with sedanolide , and counteracting protective effects of sedanolide against DSS-induced colitis were observed, which verified the significant function of FXR in the beneficial effects of sedanolide on colitis mice. Generally, sedanolide decreased the abundance of BSH-expressing bacteria which were increased by DSS administration, thus changing the composition of BAs; specifically, sedanolide led to a higher ratio of con/unconjugated BAs, which in turn inhibited the overactivation of the FXR pathway in colitis mice. But how inhibiting FXR signaling pathway improves DSS-induced colitis remains unclear. It attracted our attention that the downstream genes of FXR (SMPD3 and SMPD4) were up-regulated by sedanolide, which are reported associated with ceramide synthesis in the gut . Ceramides, a class of sphingolipids, are bioactive lipid molecules related to several vital cellular pathways, including oxidative stress, inflammation and apoptosis . The results have shown that ceramides especially ceramide (d18:1/16:0) were decreased by DSS while increased by sedanolide. The following discoveries demonstrate the role of ceramide (d18:1/16:0) in processes such as protecting cells from LPS-induced injury, diminishing cellular inflammation and enhancing the epithelial barrier, all of which are consistent with the effects of sedanolide on DSS-induced colitis mice. Previous researches have indicated the important role of ceramide in colitis that DSS induced more severe colitis in the ceramide synthase 6-deficient mice (with a reduced ability to synthesize C16-ceramide) . It was also reported that the binding of ceramide to leukocyte mono-immunoglobulin-like receptor 3 (LMIR3) could alleviate DSS-induced colitis, suggesting a mechanism of the protective effects of ceramide . Therefore, we believe that ceramide (d18:1/16:0) is the key molecule that exerts the protective effects of sedanolide on DSS-treated mice as a downstream product of the FXR-SMPD3 pathway. In summary, our study illustrated the protective effects of the natural product sedanolide against DSS-induced colitis. Compared to other potential treatments identified by researchers, sedanolide is not only a safe and bioactive natural product but also has marked effects on ameliorating inflammation, strengthening the weakened epithelial barrier and remodeling the imbalanced gut microbiota. In addition, sedanolide was reported to protect against liver injury by modulating the antioxidative Nrf2 pathway, which indicated that it may be useful for treating multiple organs and the whole body. Many natural medicines have low oral bioavailability, and how natural products exert their bioactive effects has been a question for researchers. With the rapid development of microbiomics, researchers have realized that gut microbes are closely associated with health and that any alterations in the structure or function of the intestinal flora can affect the homeostasis of the body through various signaling pathways . Using microbiomics, transcriptomics and metabolomics, we investigated the pharmacological effects of sedanolide and the mechanisms. Our study revealed the relationships among natural products, gut microbes and the host and elucidated the molecular mechanism of gut microbes. This study has several limitations. First, according to a previous study, the FXR signaling pathway was inhibited by DSS administration , which seems contrary to our findings. While our study revealed a significantly reduced ratio of conjugated BAs to unconjugated BAs, which may explain the activation of the FXR pathway after DSS administration. Second, Gonzalez et al. found that the production of ceramides in the gut was positively associated with the activation of FXR , while the transcriptome analysis and q-PCR results in our study showed activation of the SMPD3 gene in sedanolide-treated colitis mice, which indicated that another regulatory mechanism may exist for FXR-SMPD3. However, we have not identified the specific underlying mechanisms involved. Third, there were a variety of lipids that are changed after sedanolide in DSS-treated mice, but we focus on the role of ceramides; therefore, we did not perform research on other lipids that deserve deeper investigation. 

In conclusion, we found that sedanolide could alleviate DSS-induced colitis in mice in a dose-dependent manner by inhibiting inflammation and strengthening the disrupted intestinal barrier. Mechanistically, sedanolide decreased the abundances of BSH-expressing bacteria and reshaped the ratio of conjugated BAs to unconjugated BAs, thereby inhibiting the intestinal FXR-SMPD3 pathway. Furthermore, the increase in the key effector ceramide (d18:1/16:0) induced by SMPD3 ameliorated inflammation and repaired the weakened gut barrier. Our study suggests that sedanolide is a potential treatment for colitis. In addition, we identified the key lipid ceramide (d18:1/16:0), which mediates the protective effects of sedanolide, suggesting that this compound is a new research focus for curing colitis. Notably, these results provide new evidence for the analysis of the relationships among natural active molecules, intestinal microbes and hosts, and provide ideas for the study of the efficacy of natural products and its mechanism to further clarify the function of gut microbes. 

Shengjie Li: Data curation, Writing - original draft, Visualization, Software. Aoxiang Zhuge: Writing - review & editing. Hui Chen: Investigation, Validation. Shengyi Han: Methodology. Jian Shen: Methodology. Kaicen Wang: Methodology. Jiafeng Xia: Methodology. He Xia: Methodology. Shiman Jiang: Methodology. Youhe Wu: Methodology. Lanjuan Li: Supervision, Resources. 

All Institutional and National Guidelines for the care and use of animals (fisheries) were followed. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

