Introduction
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic intestinal inflammatory condition with a global prevalence [ 1 ]. In China, UC is more prevalent than CD, and the overall incidence is relatively lower compared to other countries [ 2 ]. Current treatments for IBD, mainly involving biologics and immunomodulators, often face limitations due to variable efficacy and long-term safety concerns, including an elevated risk of malignancies [ 3 , 4 ]. Therefore, exploring the pathogenesis of IBD is important for finding novel targets and developing new treatment strategies.
Traditional Chinese Medicine (TCM) adopts a holistic and integrative approach, combining diverse therapeutic modalities, including oral administration of Chinese herbal formulas, acupuncture, and other traditional techniques, to achieve multi-dimensional therapeutic effects [ 5 ]. Grounded in the principles of holistic health and individualized treatment through syndrome differentiation, TCM harnesses the synergistic properties of various medicines to manage UC [ 6 ]. This integrative approach not only aims to enhance patients’ quality of life but also seeks to mitigate the adverse effects commonly associated with conventional Western medical treatments, offering a complementary and potentially safer alternative for UC management.
Brevilin A (Br) is extracted from Centipeda minima , belonging to the Centipeda genus of the Compositae family. This traditional Chinese medicine has a history of thousands of years and is usually used to treat rhinitis, sinusitis, pain, swelling and other diseases [ 7 ]. Previous studies have demonstrated that Br has anti-cancer effects in various cancers, including multiple myeloma, breast cancer, and colon cancer [ 7 ]. It has been found that Br is a selective inhibitor of the JAK-STAT signaling pathway, which can block the JH1 domain of JAK tyrosine kinase to weaken the activity of JAKs, and can also block the STAT3 signaling in tumor cells [ 8 , 9 ]. Currently, Br is gradually recognized to have anti-inflammatory effects, such as in pneumonia, rheumatoid arthritis, and other diseases [ 10 ]. However, its efficacy in IBD remains to be explored.
In this research, we systematically explored the anti-inflammatory efficacy of Br in DSS-induced colitis. Our findings revealed that the anti-inflammatory properties of Br were primarily attributed to its modulation of the gut microbiota composition and the regulation of microbial metabolites. Additionally, we demonstrated that tumor necrosis factor-alpha (TNF-α) served as a crucial mediator in the anti-inflammatory pathway of Br. These results not only elucidate a previously unrecognized mechanism of Br’s anti-inflammatory action but also identify potential therapeutic targets for the treatment of IBD.
Methods
IBD patients samples
In this study, we obtained biopsy tissues from 15 IBD patients through colonoscopy at the First Affiliated Hospital of Nanchang University. All participants provided written informed consent, and the study received ethical approval from the hospital’s Ethics Committee ((2024)CDYFYYLK(07–061)). The collected tissues were analyzed using an immunofluorescence (IF) assay to quantify the expression levels of TNF-α proteins.
RNA-sequencing analysis
Total RNA was isolated from mouse colonic tissues using the TRIzol reagent (Invitrogen), and RNA quality was carefully evaluated prior to subsequent analyses. Biological replicates were pooled into three groups, each consisting of four samples. Strand-specific libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit and sequenced on the Illumina HiSeq X Ten platform by Genergy Biotechnology Co., Ltd. Transcript expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM) according to a previously established protocol, with Perl scripts employed for the calculations.
Differentially expressed transcripts (DETs) and functional and pathway enrichment analyses were conducted as described in our previous study [ 11 ]. Pathways were considered statistically significant when they had a P -value less than 0.05 and contained at least two associated genes.
Cell culture
HEK293T cells (obtained from ATCC, USA, RRID: CVCL_0063) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS). The cells were cultured under standard conditions of 37 °C in a humidified incubator with a 5% CO₂ atmosphere. To assess the direct anti-inflammatory effect of Br, HEK293T cells were stimulated with 1 µg/ml lipopolysaccharide (LPS) for 24 h.
Western blot
The Western blot procedure followed a similar protocol to our earlier study [ 12 ]. We used primary antibodies against the following proteins: IL-18 (cat.no.10663-1-AP, 1:1000, Proteintech, RRID: AB_2877958), IL-1β (cat.no.16806-1-AP, 1:1000, Proteintech, RRID: AB_2877959), Zo-1 (cat.no.33–9100, 1:1000, Thermo Fisher Scientific, RRID: AB_2533147), GAPDH (cat.no. 60004-1-Ig, 1:1000, Proteintech, RRID: AB_11182290), TNF-α (cat.no.17590-1-AP, 1:1000, Proteintech, RRID: AB_2881679), and β-actin (cat.no.81115-1-RR, 1:1000, Proteintech, RRID: AB_2861930). Secondary antibodies were sourced from Proteintech (Wuhan, China).
Immunohistochemistry (IHC) assay
For the identification of specific proteins in tissue samples, an IHC assay was conducted. Tissue sections were incubated with primary antibodies overnight at 4 °C, followed by a 1-hour incubation with secondary antibodies at room temperature. The target proteins were visualized using 3,3’-Diaminobenzidine (DAB) reagent (Solarbio, Beijing), and nuclei were counterstained with hematoxylin. The IHC results were evaluated by two independent pathologists, who assigned scores based on staining intensity (ranging from 0 to 3) and the percentage of positively stained cells (scored 0 to 3, with cut-off values at 0%, 30%, 60%, and 90%). The final scores were then categorized into three groups based on previous study [ 11 ].
Immunofluorescence (IF) assay
Mouse tissues were mounted on glass slides and incubated for 24 h. Subsequently, the tissues were fixed with 4% paraformaldehyde for 15–20 min, followed by permeabilization with 0.1% Triton X-100 for 1 h. The tissues were then incubated overnight at 4 °C with primary antibodies, including Zo-1 (cat.no.33–9100, 1:1000 dilution, Thermo Fisher Scientific) and TNF-α (cat.no.17590-1-AP, 1:1000 dilution, Proteintech). After washing, the tissues were incubated with fluorescently-labeled secondary antibodies, and the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). The immunofluorescence signals were visualized and quantitatively analyzed using confocal microscopy, with excitation wavelengths set at 488 nm and 594 nm to detect the specific fluorescent dyes conjugated to the secondary antibodies.
Establishment and identify of DSS-induced colitis model in mice
DSS-induced acute colitis model was established based on a previously published protocol [ 11 , 13 ]. Male C57BL/6 mice (aged 6–8 weeks) were procured from Wuhan Beisai Model Biotechnology Co., Ltd. (RRID: IMSR_JAX:000664). DSS from MP Bio was used to induce colitis, while Br was obtained from RENI Pharmaceutical Technology Co., Ltd. (Sichuan, China). Prior to the experiment, all mice underwent a one-week acclimation period. Subsequently, the mice were randomly allocated into two groups: the DSS group and the DSS + Br (20 mg/kg) group. To induce colitis, mice were provided with drinking water containing 3% DSS for 9 consecutive days. In the DSS + Br group, oral gavage administration of Br (20 mg/kg) commenced on day 0. The dose of Br (20 mg/kg) was selected based on its established efficacy and safety profile in previous in vivo studies of colorectal cancer and other neoplastic diseases [ 7 , 14 , 15 ]. Throughout the modeling process, body weight fluctuations and hematochezia were closely monitored. On day 10, the mice were humanely euthanized, and serum and colon tissue samples were collected for subsequent analyses.
For the microbiota-depleted mice model, intestinal decontamination was initiated when the mice reached 4 weeks of age. A combination of antibiotics was administered for approximately two weeks, as described in previous literature [ 11 , 16 ]. For fecal microbiota transplantation, microbiota samples (derived from donor mice treated with Br) were resuspended in a 30% glycerol/PBS solution alongside phosphate-buffered saline (PBS) controls. Following centrifugation, the concentration was adjusted to an optical density at 600 nm (OD600) of 1.0, and the samples were stored at −80 °C. Microbiota-depleted mice were then randomly divided into two groups: one group received microbiota transplantation, while the other served as a control group receiving PBS.
Histological analysis of colitis
Colon tissues were processed as previously described [ 17 ]. Specifically, the tissues were fixed in 4% formaldehyde solution for 24 h and then embedded in paraffin. Subsequently, paraffin sections were prepared and stained with hematoxylin and eosin (HE). The stained sections were examined under a microscope to assess the degree of inflammation. The severity of inflammation was evaluated using the grading system established by Truelove and Richards [ 18 ].
ELISA assay
Mouse serum levels of IL-1β and TNF-α were quantified using respective ELISA kits. The experimental procedure was as follows: First, mouse serum samples were diluted threefold. Then, 100 µL of the standard solutions and diluted samples were added to the designated wells of the microplate. The plate was covered and incubated at 37 °C for 90 min. After decanting the liquid from the wells, 100 µL of the working solution was added, followed by a 1-hour incubation at 37 °C. Subsequently, the wells were washed, and 100 µL of the HRP-conjugated working solution was added. This was incubated at 37 °C for 30 min. Finally, the stop solution was added, and the optical density was measured at 450 nm.
16S rRNA amplicon sequencing
Fecal DNA was extracted by Shanghai OE Biotech. Co., Ltd [ 11 ]. The extraction process involved sample lysis, Proteinase K treatment, and impurity removal. The concentration and integrity of the extracted DNA were verified using a NanoDrop 2000 spectrophotometer and agarose gel electrophoresis, respectively. The V3-V4 hypervariable regions of the 16 S rRNA gene were then amplified via PCR using universal primers 343 F (5’-TACGGRAGGCAGCAG-3’) and 798R (5’-AGGGTATCTAATCCT-3’)) [ 11 ]. After assessing the amplicon quality, the PCR products were purified with AMPure XP beads (Agencourt). Subsequent steps, including the construction of final amplicons and alpha/beta diversity analyses, were also performed by Shanghai OE Biotech. Co., Ltd., following their standard protocols.
Liquid chromatography tandem-mass spectrometry (LC-MS) analysis
The experimental procedure followed a previously published protocol [ 11 ]. Simply, samples were mixed with L-2-chlorophenylalanine, methanol, and acetonitrile, vortexed, and sonicated in an ice bath for 10 min. The supernatant was collected, filtered, and transferred to LC vials for LC-MS analysis. Shanghai Luming Biological Technology Co., Ltd. analyzed the metabolomic data using Progenesis QI software (V2.3) software for baseline filtering, peak processing, and data normalization. Compounds were identified by m/z, fragments, and isotopic patterns with reference to HMDB. Peaks with missing values in over 50% of groups were removed. Principal component analysis (PCA) in R was used to check sample distribution and analytical stability.
Statistical analysis
Statistical analyses were conducted using GraphPad Prism 8 and R software. To assess the significance of metabolite differences between two groups, a two - tailed Student’s t-test was employed. To compare the differences in TNF-α expression in patient samples, we used non-parametric tests (Mann-Whitney U test), while for other experiments, we primarily used paired t-tests as they were paired experiments. For comparisons among three or more groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used. Each experiment was replicated at least twice, and statistical significance was defined as a P -value less than 0.05.
Results
Br mitigates mouse colitis induced by DSS
To investigate the anti-inflammatory effects of Br on colitis, acute mouse colitis was induced by administering 3% DSS for nine days. As illustrated in Fig. 1 A, the mice were randomly allocated into two groups: the DSS + PBS group and the DSS + Br group. From day 1 onwards, mice in the DSS + Br group received oral gavage of Br at a dose of 20 mg/kg every other day. Compared with the DSS + PBS group, mice treated with Br showed significantly increased colon length and body weight (Fig. 1 B and D). Nevertheless, these values remained lower than those of the control group. Histological examination demonstrated that the colons of mice in the DSS + PBS group suffered more severe structural damage and had a greater infiltration of inflammatory cells than those in the DSS + Br group (Fig. 1 E). Additionally, the level of protein indicated that cell viability and barrier integrity were compromised in the DSS + PBS group, as evidenced by lower expressions of Ki67 and Zo-1 compared to the Br-treated group (Fig. 1 F).
Fig. 1 Effect of Brevilin A (Br) on dextran sulfate sodium (DSS)-induced colitis mouse model in vivo. A-B The methods of medicine irrigation and representative image of colons from mice model. C The statistical results of colon length among the three groups (* P < 0.05, ** P < 0.01 vs. DSS group). D Body weight of mice in DSS treatment and control groups was determined once per day from 0 to 10th day before and after DSS administration and Br administration (* P < 0.05, ** P < 0.01 vs. DSS group). E Macroscopic changes and histological score in the colons of mice and the score of disease activity from different mouse (scale bar, 200X) (* P < 0.05, ** P < 0.01 vs. DSS group). F The protein levels of Ki67 and Zo-1 were examined by IHC and IF assay, respectively (scale bar, 200X, respectively), the right pictures were statistical results (* P < 0.05, ** P < 0.01 vs. DSS group). Data are presented as mean ± SEM, n = 4 mice per group. A probability value of less than 0.05 ( P < 0.05) was adopted as the threshold for establishing statistical significance
Br exerts its anti-colitis effects through the TNF-α signaling pathway and by modulating pro-inflammatory factors
RNA sequencing analysis of bowel tissue from mice was conducted to comprehend the potential mechanisms underlying Br’s effects on colitis development (Fig. 2 ). Among DSS + PBS, control and DSS + Br groups, the expressed genes are obviously different according to the PCA analysis (Fig. 2 A and B). Among the differentially expressed genes (DEGs), proinflammatory factors such as IL1β, IL1α, Cxcl3, Cxcl2, and TNF-α were identified in the heatmap, indicating the overexpression of these genes in colitis tissue induced by DSS (Fig. 2 C and D). Wikipathway analysis unveiled significant variations in the inflammatory response pathway, comprehensive IL-17 A signaling and TNF signaling pathway (Fig. 2 E and F). Further GO enrichment analysis demonstrated that DEGs were associated with immune response signaling pathways and inflammation response to lipopolysaccharide (LPS) (Fig. 2 G), suggesting a connection between Br’s anti-inflammatory mechanism and function of immune regulation. We performed the co-interactive function analysis among the DEGs, finding that the members of the TNF-α family, such as TNFrsf11b and TNFsf11, interacted with other DEGs in the development of colitis (Fig. 2 H and I). To substantiate this hypothesis, the levels of IL-1β and TNF-α in the serum were measured in mice, revealing lower levels in the DSS + Br group than in the DSS + PBS group, as well as the protein level which was assessed by western blot and IHC assays (Fig. 3 A and C). Moreover, bowel tissues from Crohn’s disease (CD) patients were collected for immunofluorescence assays to measure TNF-α protein expression. The results indicated higher TNF-α protein expression in IBD tissues (Fig. 3 D). In alignment with the above findings, to induce inflammatory response of HEK293T cells, after being stimulating with LPS (1 µg/ml), we treated 20 µg/ml Br in a time-dependent manner on HEK293T cells, finding that Br inhibits IL18, IL-1β, and TNF-α as the increasing of time; however, it seemed that the level of IL-1β and TNF-α did not decrease as the concentration of Br increased (Fig. 3 E). To explore the impact of Br on TNF-α in a mouse model, we established a colitis model and administered Br or TNF-α adeno-associated virus (AAV) to the mice. As outlined in the in vivo assay scheme (Fig. 4 A), Br was administered at a dosage of 20 mg/kg via gastric gavage every two days, and TNF-α AAV was delivered intraperitoneally to the mice on two occasions.
Fig. 2 Brevilin A (Br) mitigated the damage of mucosa barrier via regulating inflammatory response pathway and TNF-α signaling pathway. A-B The PCA analysis of expressed genes profile between control and Br-treated mice or between control and DSS-treated mice in colitis model. C-D Heatmap showed differentiated expressed genes profile between control and Br-treated mice or between control and DSS-treated mice in colitis model. E-F KEGG Enrichment analysis of differential genes among the three groups. G GO Enrichment Analysis of differential genes among the three groups. H-I protein-protein interaction network was performed using the R software tool. Data are presented as the mean ± SEM. (* P < 0.05, ** P < 0.01 vs. DSS group)
Fig. 3 The expression of TNF-α in mouse colitis model and IBD tissue. A The statistical results and expression of IL-1β and TNF-α in serum of mice by ELISA assay. B-C Expression of TNF-α in colon tissue were measured by western blot assay and IHC assay (scale bar, 200X) (* P < 0.05, ** P < 0.01 vs. DSS group). D The level of TNF-α in the colon tissue of IBD patients was measured by IF assay (scale bar, 200X) (* P < 0.05, ** P < 0.01 vs. DSS group). E The level of TNF-α, IL18 and IL-1β in the colitis tissue of mice were measured by western blot assay. Data are presented as the mean ± SEM
Fig. 4 Effects of TNF-α aggravated DSS-induced colitis in the Brevilin A (Br)-treated mice. A Schematic description of the animal experimental design. After 1 weeks of rest, mice were randomly divided into four groups: DSS group, DSS + Br group, DSS + Br + TNF-α virus and DSS + TNF-α virus. B-D Represent colon imaging, comparison of colon length and Body weight change of each group (* P < 0.05, ** P < 0.01 vs. DSS group). E-F The IL1β and TNF-α level in the serum were measured by ELISA assays (* P < 0.05, ** P < 0.01 vs. DSS group). G-H Representative images of H&E staining of colon sections (scale bar, 200X) and scores were judged according to scoring criteria in these groups (* P < 0.05, ** P < 0.01 vs. DSS group). I The protein levels of TNF-α and Zo-1 were examined by Western blot assay. Data are presented as mean ± SEM, n = 4 mice per group
Regarding colon length and body weight, mice treated with TNF-α AAV showed significant reductions compared to the DSS group (Fig. 4 B and D). In contrast, both the Br group and the Br + TNF-α AAV group showed increases in colon length and body weight. The levels of inflammatory cytokines, including IL-1β and TNF-α, were highest in the TNF-α AAV group, decreased in the Br + TNF-α AAV group, and were lowest in the Br group (Fig. 4 E and F).
Histological analysis revealed more severe structural damage and inflammatory cell infiltration in the colonic tissues of the DSS and TNF-α AAV groups than in the Br + TNF-α AAV group. The Br group showed the least severe pathology (Fig. 4 G and H). Furthermore, we demonstrated that the expression of Zo-1 was increased while TNF-α protein levels were decreased in the intestinal tissues of Br-treated mice compared to the DSS and TNF-α AAV groups (Fig. 4 I).
Histological analysis revealed that colonic tissues from the DSS and TNF-α AAV groups displayed more severe structural damage and infiltration of inflammatory cells than those in the Br + TNF-α AAV group, while the Br group showed the least severe pathology (Fig. 4 G and H). Furthermore, we demonstrated that the expression of Zo-1 was increased while TNF-α protein levels were decreased in the intestinal tissues of Br-treated mice compared to the DSS and TNF-α AAV groups (Fig. 4 I).
The gut microbiota plays a role in br’s anti-colitis effects
We used 16 S rRNA sequencing to analyze the impact of Br on the gut microbiota (Fig. 5 ). Br treatment significantly increased the α-diversity of the gut microbiota (Fig. 5 A and B). PCA demonstrated the significant restructuring of gut microbiota by Br (Fig. 5 C). Notably, bacterial compositions underwent significant alterations due to Br treatment (Fig. 5 D). A heatmap of the top 15 bacterial species showed that Bacteroides vulgatus , Escherichia coli , Clostridium perfringens , Lactobacillus murinus , Paeniclostridium sordellii , Bacteroides thetaiotaomicron , Bacteroides caecimuris , and Romboutsia ilealis were enriched in DSS-treated mice. In contrast, Lactobacillus johnsonii , Burkholderiales bacterium , and Ileibacterium valens were predominantly enriched in the control and Br-treated groups (Fig. 5 E). Furthermore, co-occurrence network analysis revealed compelling co-exclusion relationships between beneficial and pathogenic bacteria in Br-treated mice, suggesting that Br-enriched beneficial bacteria may inhibit pathogens, thereby alleviating colitis (Fig. 5 F). KEGG analysis revealed that these differentiated expressed bacteria were implicated in material metabolism processes such as lipid metabolism, carbohydrate metabolism, and amino acid metabolism, among others (Fig. 5 G).
Fig. 5 Brevilin A (Br) alters gut microbiota in the stool. A-B Shannon index and Simpson index of the gut microbiota in three groups mouse colitis model. C Stool micro biotic profile significantly differed among control group, Br-treated and single DSS mice by principal component analysis (PCOA). Comparisons of α- and β-diversity were accessed by 2-tailed Mann-Whitney U test and permutational multivariate analysis of variance, respectively. D Bar chart for the gut microbiome of three groups. Differences in abundance were detected by using a multivariate statistical model. E heatmap for the top 15th microbiome of the three groups. F Ecological network among differential bacteria in the three groups. G The heat plot showed the associated metabolism pathways that were involved in differential abundance of gut microbiome. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01 vs. DSS group
To investigate whether the gut microbiota mediates the therapeutic effects of Br, we administered an antibiotic cocktail to deplete the gut microbiota in C57BL/6 mice. These microbiota-depleted mice then received a fecal microbiota transplantation (FMT) from donor mice that had been treated with either Br or PBS (without DSS treatment). The experimental timeline is outlined in Fig. 6 A. Notably, mice that received FMT from Br-treated donors (FMT-Br) exhibited a significant increase in colon length and body weight compared to those that received FMT from PBS-treated donors (FMT-PBS) (Fig. 6 B and C). Consistent with this, histological analysis of colon tissue by HE staining revealed a more pronounced alleviation of colitis in the FMT-Br group than in the FMT-PBS group (Fig. 6 D). Furthermore, immunohistochemical analysis showed that the FMT-Br group maintained higher expression levels of the proliferation marker Ki67 and the tight junction protein Zo-1, along with a lower level of TNF-α, in the colonic mucosa compared to the FMT-PBS group (Fig. 6 E). Collectively, these findings demonstrate that the protective effects of Br against colitis can be transferred via the gut microbiota, highlighting its crucial role in mediating the therapeutic action of Br.
Fig. 6 The gut microbiota from Brevilin A (Br)-treated mice alleviated DSS-induced colitis. A Schematic description of the animal experimental design. After two weeks of intestinal sterilization treatment, mice were randomly divided into two groups: DSS group, DSS + Br group. B-C The statistical analysis of colon length and body weight in the mice receiving flora enema from vehicle control (PBS) or Br, respectively. D-E Representative HE assays, IF and IHC images showing colitis, Ki67, Zo-1 and TNF-αexpression in the two groups, respectively. The statistical analysis was on the right. Data are presented as mean ± SEM, n = 4 mice per group.* P < 0.05, ** P < 0.01 vs. Control group
Microbiota metabolism is potent essential to anti-inflammation effects of Br
For the purpose of identifying potential molecules, LC-MS was conducted using feces from mice treated with DSS + Br, DSS + PBS and control mice. The top 50 metabolites ( P < 0.05) including including 7-Oxolithocholic Acid, Kudinoside A, Veratrine and Soyasaponin, and so on, exhibited significant alterations in the DSS + Br and DSS + PBS groups compared to the control group (Fig. 7 A and C). Among these metabolites, KEGG pathway analysis revealed enrichment in purine metabolism, ABC transporters, phenylalanine, tyrosine, and tryptophan biosynthesis, and oxidative phosphorylation, among others (Fig. 7 B and D). Further Reactome analysis indicated that the metabolites were implicated in the signaling of GPCR and the DNA damage pathway (Fig. 8 A and B). Notably, aminoacyl-tRNA biosynthesis, riboflavin metabolism, and central carbon metabolism in cancer were also involved (Fig. 8 ). Additionally, the differentially expressed metabolisms associated with aminoacyl-tRNA biosynthesis included L-arginine, L-proline, L-tyrosine, and L-lysine, among others (Fig. 8 C and E). Concerning riboflavin metabolism, four metabolites, including flavin mononucleotide, lumichrome, riboflavin, dimethylbenzimidazole, and 1-Deoxy-L-Glycero-Tetrulose4-phophate, were involved (Fig. 8 F and H). Nevertheless, the majority of differentially expressed metabolisms were involved in the central carbon metabolism in cancer, such as L-serine, fumaric acid, and L-cysteine, among others (Fig. 8 I and K).
Fig. 7 Brevilin A (Br) alters gut microbiota–related metabolites in the stool. A Stool metabolic profile significantly differed between control and DSS-treated mice showed by Volcano Plot and heat map. B KEGG Enrichment analysis of differential metabolites between control and DSS-treated mice. C Stool metabolic profile significantly differed between Br + DSS treated and DSS-treated mice showed by Volcano Plot and heat map. D KEGG Enrichment analysis of differential metabolites between control and DSS-treated mice
Fig. 8 The signaling pathways that involved in the gut microbiota–related metabolites. A-B Reactome analysis of differential metabolites among control, DSS-treated and Brevilin A (Br) + DSS treated mice. C-E Aminoacyl-tRNA biosynthesis was found to be associated with differentiated expressed metabolites by Gene Set Enrichment Analysis (GSEA) among the three groups. F-H Riboflavin metabolism was found to be associated with differentiated expressed metabolites by GSEA among the three groups. I-K Central carbon metabolism in cancer was found to be associated with differentiated expressed metabolites by GSEA among the three groups
Discussion
IBD, characterized by its recurrent nature, resistance to cure, and potential to progress into cancer, has emerged as a substantial and persistent threat to human health and well-being [ 19 ]. Given the significant challenges posed by IBD, there is an urgent need to develop treatments that are affordable, effective, and sustainable. Traditional Chinese Medicine (TCM), with its inherent advantages of multi-targeted approaches and relatively low side effects, has garnered substantial interest for its potential application in treating such complex chronic inflammatory diseases [ 5 ]. In our research, we have identified Br—an active compound derived from the natural product Centipeda minima—as a promising candidate for enhancing intestinal health. Specifically, Br has been shown to mitigate the progression of colitis in a mouse model. Furthermore, the therapeutic mechanism of Br seems to be intricately associated with the modulation of inflammatory cytokine secretion and the restoration of gut microbiota equilibrium.
TNF-α, a cytokine predominantly secreted by activated macrophages, natural killer (NK) cells, and other immune cells, exerts a pivotal function in orchestrating inflammatory responses [ 20 ]. Abundant evidence indicates that the overproduction of TNF-α represents a key driver in the pathogenesis of IBD [ 21 ]. In our study, we identified TNF-α signaling pathway was potent target of Br in terms of its anti-inflammation function. Furthermore, two genes associated with the TNF superfamily, Tnfsf11 and Tnfrsf11b, were found to interact with other genes.
TNF-α mediates its biological actions by binding to either TNF-receptor 1 (TNFR1) or TNF-receptor 2 (TNFR2), initiating distinct intracellular signaling cascades [ 20 ]. When TNF-α binds to TNFR1, it commonly elicits pro-inflammatory responses and promotes apoptosis. In contrast, binding to TNFR2 predominantly activates anti-inflammatory mechanisms and cell survival pathways [ 20 ]. IL1α and IL1β were co-interacted according to the protein-protein interaction network. Furthermore, by comparing transcriptomes from the inflamed colonic tissue of DSS-treated mice and the intestinal tissue of Br-treated mice, we found DEGs were involved in the inflammatory response pathway, IL17-pathway and TGF-β signaling pathway. All these signaling pathway are associated with the development of inflammation, suggesting Br exerts its anti-inflammatory effects by modulating these inflammatory signaling pathways. Previous studies have demonstrated that Br can attenuate dermatitis by inhibiting inflammation and Th17 cell differentiation, also, it can alleviate acute lung injury through inhibiting the NF-κB signaling pathway [ 9 , 22 ]. Some previous studies have utilized 293 T cells to investigate protein changes related to cellular inflammation induction, and we conducted part of our experiments based on their methodologies [ 23 , 24 ]. Admittedly, this approach still has its limitations. Importantly, our in vivo data from DSS-induced colitis models and human IBD tissues strongly support the role of Br in modulating TNF-α signaling and gut microbiota.
Moreover, emerging research has shown that the intestinal microbiome of IBD patients undergoes profound changes, marked by decreased richness and diversity [ 25 ]. Specifically, individuals with IBD typically exhibit elevated levels of Bacteroides , a genus of bacteria that can disrupt the intestinal barrier and instigate colonic inflammation. Concurrently, there is a notable reduction in the levels and diversity of mucosal Lactobacillus species [ 26 ]. Given the crucial role of the gut microbiota in health and disease, including its ability to metabolize drugs and maintain intestinal homeostasis [ 27 ]. Considering the pivotal role of the gut microbiota in these processes, we utilized 16 S rRNA sequencing to evaluate its diversity. This approach aimed to uncover how Br influences the gut microbiota during colitis development, providing insights into the underlying mechanisms of its therapeutic effects.
As for the colitis inflammation, Proteobacteria , Firmicutes , and Bacteroidota are predominantly present and are considered indicators of microbial instability, particularly in the bowel of mice with colitis [ 28 ]. Specifically, our findings demonstrated that Br could significantly augment the relative abundance of beneficial gut microbes such as Burkholderiales bacterium , Ileibacterium valens , and Lactobacillus johnsonii , while decreasing the relative abundance of pathogenic or pro-inflammatory microbes like Bacteroides vulgatus , Escherichia coli , and Clostridium perfringens . This shift in microbial composition effectively alleviated gut microbiota dysbiosis in mice with colitis.
The adhesion of microbes and alterations in microbial interactions with intestinal epithelial cells are key factors in activating inflammatory cytokines in patients [ 29 ]. In this study, pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-22, were significantly elevated in DSS-treated mice but were notably reduced in Br-treated mice. This reduction in cytokine levels was correlated with the changes in microbial abundance.
Lactobacillus , a highly recognized probiotic genus, is pivotal in safeguarding intestinal barrier function and mucosal immunity [ 30 , 31 ]. Burkholderiales bacterium , a representative of the Proteobacteria phylum, has been observed to inhabit and become a dominant component in the gut microbiota of patients with active IBD, although its exact mechanism remains unclear [ 32 ]. Conversely, the symbiotic bacterium Bacteroides vulgatus has been shown to exacerbate colitis symptoms in mice when present [ 33 ].
Furthermore, recent studies have highlighted that intestinal bacteria can regulate immunity and inflammation by altering bacterial metabolites in the bowel. In our research, we employed LC-MS to profile the metabolites in a Br-treated colitis mouse model. Over a hundred compounds were identified or tentatively characterized, including 7-Oxolithocholic Acid, Kudinoside A, Veratrine, and Soyasaponin. Notably, these metabolites exhibit a wide range of biological activities, such as involvement in GPCR signaling, the DNA damage pathway, aminoacyl-tRNA biosynthesis, riboflavin metabolism, and central carbon metabolism in cancer [ 34 – 36 ].
However, the therapeutic potential of these metabolites, either individually or in combination, for the treatment of IBD remains to be fully elucidated. A limitation of this study is the use of a single dose of Br. Future investigations should include comprehensive dose-response studies and pharmacokinetic analyses to determine the optimal dosing regimen and to understand the absorption, distribution, and metabolism of Br in the context of colitis. Therefore, in the future, we aim to uncover the pharmacodynamic substances among these metabolites and explore their potential mechanisms of action in the treatment of colitis.
Conclusions
Preventive administration of Br alleviated symptoms in DSS-induced mice, while its therapeutic use significantly reduced inflammatory mediators, which was linked to the TNF-α signaling pathway and changes in gut microbiota such as the decline of harmful bacteria, the increase of beneficial bacteria, and metabolic alterations. This study lays a foundation for exploring the therapeutic features of Br, showing great promise for innovating IBD treatments.
Abbreviations
Inflammatory bowel disease
Traditional chinese medicine
Immunohistochemistry
Immunofluorescence
Liquid chromatography tandem-mass spectrometry
Dextran sulfate sodium
Ulcerative colitis
Crohn’s disease
Tumor necrosis factor-alpha
Principal component analysis
Differentially expressed genes
Brevilin A
Publisher’s note
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LimingChen and ChaotaoTang have contributed equally to this work.
Acknowledgements
Not appliable.
Author contributions
Liming Chen and Chaotao Tang: experiment performing, data analysis, and manuscript writing. Diping Hu and Shujiao Yu: sample collecting and data analysis. Peng Liao: project development.
Funding
The project supported by Middle-aged Backbone Talents in the Province (grant No. 2024000003-09, PI: Peng Liao).
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
Our study was permitted by Ethics Committee of the First Affiliated Hospital of Nanchang University. The ethics approval ID is (2024)CDYFYYLK(07–061).
Consent for publication
The authors agreed.
Competing interests
The authors declare no competing interests.
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