Trans-Anethole Alleviates DSS-Induced Ulcerative Colitis by Remodeling the Intestinal Flora to Regulate Immunity and Bile Acid Metabolism

Ulcerative colitis (UC) is the most common inflammatory bowel disease (IBD); it is incurable, and the treatment is expensive. Trans-anethole (TA), the main component of fennel, exhibits various biological activities. An increasing number of studies have demonstrated the efficacy of herbal active ingredients in the treatment of UC. This study aimed to investigate the effect and mechanism of TA in UC. In this study, we have experimented on mice with dextran sulfate sodium salt (DSS)-induced UC. The TA group was gavaged with 62.5 mg/kg TA by gavage once daily on days 8–14. To observe the effect of TA on the colon tissue, various investigations were performed, including western blot and immunohistochemistry for intestinal barrier protein expression, TUNEL staining for apoptosis, western blot, and ELISA for inflammation level, flow cytometry for Th17/Treg, LC–MS for blood bile acid content, GC–MS for blood fatty acid content, and 16s RNA for intestinal contents. TA alleviated weight loss in mice with UC; increased colon length; alleviated intestinal mucosal damage; upregulated claudin-1, occludin, and ZO-1 protein expression levels; reduced inflammatory factors in the colon and serum; and alleviated apoptosis. TA reduced fatty acid and bile acid levels by inhibiting colony abundance and reducing Th17/Treg cell differentiation in the colon. We found that TA alleviates DSS-induced UC by remodeling the intestinal flora to regulate immunity and bile acid metabolism.


Introduction
Colitis is an inflammatory lesion of the inner layer of the colonic mucosa; there are several types of colitis [1].The pathological process of colitis is unclear, and its pathogenesis involves genetic and immune-mediated causes, infectious agents, drugs, and bile acid malabsorption [2][3][4].Ulcerative colitis (UC) is a common chronic form of colitis that has a long course, which is difficult to treat [5].The clinical manifestations of UC include abdominal pain, mucus production, diarrhea, and bloody stools [6].The incidence and prevalence of UC are continue to increase, with North America and Europe having the highest prevalence [7].The inflammation in UC is mainly concentrated in the colon (95%), and the terminal ileum is also affected in some patients, making it indistinguishable from Crohn's disease [8,9].Studies have shown that abnormal responses to environmental factors, genetic susceptibility, abnormal immune regulation, intestinal mucosal barrier, and altered intestinal microecology are important factors in the development of UC [10].Various treatments are available for UC, including anti-inflammatory, immunosuppressive, and biological therapies; however, a large number of patients with UC do not benefit from these treatments because of their insensitivity or significant treatmentrelated adverse effects [11].5-Aminosalicylic acid, mesalazine, steroids, and immunosuppressants are the main drugs used to treat UC [9].Studies have found that drug therapy can cause resistance and complications, and the patients had to undergo colectomy [12].Therefore, treatment efficacy, safety, and quality of life should be considered in the future for patients with UC.
Inflammatory destruction and repair of the intestinal mucosa are key features of inflammatory bowel disease (IBD).In the gastrointestinal tract, tight junction proteins are located at the interface between the epithelial cells, which are responsible for maintaining the integrity and function of the intestinal mucosal barrier [13].The intestinal epithelial cells play a key role in maintaining homeostasis between the microbiota and the host, producing physical barriers such as mucus layers, glycocalyxes, and cell junctions and chemical barriers that subtly isolate the symbiotic microorganisms from host immune cells to prevent unnecessary conflicts between symbiotic microbes and host immune cells, thus maintaining symbiotic relationships in the gut [14].Bacterial or endotoxin invasion is primarily recognized by Toll-like receptor 4, a typical member of the pattern recognition receptor family, which recognizes bacterial components, such as lipopolysaccharides, and can trigger a signaling cascade leading to activation of NF-κB, which triggers a series of proinflammatory immune responses through the host's attempt to destroy the invading pathogen [15,16].Disruption of the mucosal barrier in patients with UC can trigger bacterial and endotoxin translocation, leading to loss of innate immunity and abnormal activation of acquired immunity [17]; thus repair of the intestinal mucosal barrier is crucial in the treatment of colitis.
In recent years, a more popular theory has suggested that dysregulation of the gut immune response to microorganisms is one of the causes of onset, progression, and changes in UC [18].The intrinsic and adaptive immunities of the host prevent the invasion of harmful bacteria and tolerate normal microbiota.However, an imbalance in the intestinal flora leads to a decrease in intestinal immune function and overstimulation of the intestinal mucosal immune response, which eventually leads to the development of IBD [19].Gut microbiota disorders, also known as ecological dysbiosis or flora imbalance, can promote susceptibility to colitisassociated tumors by overstimulating CD8 T cells, promoting chronic inflammation and early T-cell failure and thereby reducing antitumor immunity [20].One study found that gut microbes have an important role in brain function and behavior [21].Vamanu et al. [22] found that the dynamic activity of the microbiota can prevent the development of degenerative diseases through food control and consumption of new active ingredients.The composition of the three major bacterial phyla in the intestinal flora of patients with IBD was found to be disturbed, with a decreased proportion of thickwalled and anaphylactic phyla and an increased proportion of anaphyla [23].The gut microbial composition is a powerful inducer of intestinal pro-inflammatory T helper 17 cells (Th17) and regulatory T cells (Treg).Through the induction of type 3 retinoic acid-related orphan receptor γt (RORγt) (+), Tregs cells and Th17 cells regulate type 2 responses and balance mucosal immune responses [24].During UC progression, inflammation leads to a usual increase in the number of Th17 cells and a decrease in Treg that suppresses Th17 activity [25].The intestinal flora can interact with the metabolites such as bile acids (Bas) to regulate host metabolism by regulating the microbial composition of the body, mainly through the activation of immune genes in the small intestine [26,27].According to the literature, BAs play an important role in intestinal immune homeostasis by regulating immune and inflammatory processes through signaling pathways, such as the farnesoid X receptor (FXR) regulatory pathway and cell surface G protein-induced signaling [28], as well as by controlling the release of immunoglobulin A antibodies from intestinal microbes [29].One study found that short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate were able to produce bacterial species with metabolites that had positive effects on the intestinal mucosa [30].Butyrate is the main energy source of colonocytes that has antiinflammatory and intestinal homeostasis effects [31,32].Thus, the interaction between the intestinal flora, mucosal immunity, and BA metabolism plays a key role in the pathogenesis of UC.
Trans-anethole (TA), C 10 H 12 O, is the main component extracted from aniseeds, anises, and fennels.It has antiinflammatory, antioxidant, anticancer, neuroprotective, and vascular activities [33].TA ameliorates chronic lung inflammation in mice with chronic obstructive pulmonary disease [34].TA also inhibits Th2 cytokines to reduce ovalbumininduced airway inflammation [35].However, it is unknown whether anethole has anti-inflammatory and immunomodulatory effects in colitis.Herbal monomeric compounds have been developed to treat UC.Studies have shown that baicalin attenuates the Th17/Treg ratio and reduces the ratio of thickwalled to anaphylactoid phyla and the level of endotoxincontaining anaphylactoid phyla in the stool of rats with UC and can be used as a prebiotic agent for the treatment of inflammation and intestinal ecological dysregulation associated with UC [36].Berberine improves Treg/Th17 homeostasis in dextran sulfate sodium salt (DSS)-induced UC models, decreases the diversity of intestinal microorganisms, and interferes with the relative abundance of Desulfovibrio, Eubacterium, and Bacteroides [37].This demonstrates the promising potential of the active ingredients of herbal medicines for the prevention and treatment of UC.
2.10.Flow Cytometry.The isolate (TBD, LDS1090) was added to the blood and centrifuged at 400 g for 30 min.
The second layer of liquid was transferred to PBS, centrifuged at 250 g for 10 min, and the supernatant was aspirated to obtain mouse peripheral blood mononuclear cells (PBMC).
2.11.High Throughput 16S rDNA Sequencing.The DNA of the microflora was extracted, the concentration and purity of the DNA samples were checked using a Nanodrop, and the integrity of the DNA samples was checked using agarose gel electrophoresis.Specific segments of the variable region (V3-V4) were selected for PCR amplification, purification, and recovery of the amplification product using magnetic beads, fluorescence quantification of the amplification product, the fluorescence reagent Quant-it PicoGreen dsDNA Assay Kit, and a Microplate Reader (BioTek, FLx800).The Illumina TruSeq Nano DNA LT Library Prep Kit was used to prepare the sequencing libraries.Library size distribution was measured using an Agilent 2100 Bioanalyzer, and library concentration was determined by Qubit 3.0 or fluorescent quantitative PCR.The libraries were then sequenced, and the data were analyzed.
2.12.Gas Chromatography-Mass Spectrometry.Standards: acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acids were purchased from Sigma-Aldrich.Serum was added to a 2 mL centrifuge tube; 50 μL of 15% phosphoric acid, 10 μL of 75 μg/mL internal standard (isocaproic acid) solution, and 140 μL of ether were vortexed and shaken for 1 min; and the supernatant was centrifuged at 4°C for 10 min at 12,000 rpm.The supernatant was collected and analyzed using a gas chromatography-mass spectrometer (Suzhou Anyipu Precision Instrument Co., Ltd.).Chromatographic conditions: agilent HP-INNOWAX capillary column (30 m * 0.25 mm ID * 0.25 μm); split injection with a volume of 1 μ and split ratio of 10 : 1; and 250, 300, and 250°C temperatures for the injection port, iron source, and transmission line, respectively.The programed ramp-up temperature started at 90°C, followed by ramping up to 120°C at 10°C/min, 150°C at 5°C/min, and finally 250°C at 25°C/min for 2 min.Helium was used as the carrier gas at a flow rate of 1.0 mL/min.
Mass spectrometry conditions were as follows: electron bombardment ionization (EI) source, SIM scan mode, and electron energy of 70 eV.
Chromatographic conditions: agilent HP-INNOWAX capillary column (30 m * 0.25 mm ID * 0.25 μm); split injection with a volume of 1 μL and split ratio of 10 : 1; and 250, 300, and 250°C temperatures for the injection port, iron source, and transmission line, respectively.The programed ramp-up temperature started at 90°C, followed by ramping up to 120°C at 10°C/min, 150°C at 5°C/min, and finally 250°C at 25°C/min for 2 min.Helium was used as the carrier gas at a flow rate of 1.0 mL/min.Mass spectrometry conditions were as follows: electron bombardment ionization (EI) source, SIM scan mode, and electron energy of 70 eV.
2.14.Statistical Analyses.All data are presented as means AE SD.The SPSS v23.0 software was used for data analysis.One-way ANOVA was used to compare data between multiple groups, and t-test was used to compare two groups.P <0:05 was considered statistically significant.

DSS-Induced
Colitis in BALB/c Mice.HE staining at week 15 showed that DSS feeding for 11 weeks caused colonic lesions in BALB/c mice.Compared with the control group, the colon crypt in the model group was swollen and destroyed; lymphocytic infiltration, diffuse mucosal congestion, edema, granular mucosal surface, increased mucosal 4 Mediators of Inflammation fragility, and ulceration were noticed.The crypt surface was uneven, the crypt gap was enlarged, and the epithelium was damaged.After TA treatment, the mucosal layer remained intact, the crypt gap was reduced, and a U-shaped crypt was visible (Figure 1(a)-1(f )).TEM revealed damaged ileal intestinal mucosa, reduced microvilli, and incomplete mitochondria in the model group (Figure 1(b)).The body weight of mice gradually decreased after DSS feeding, and the body weights of mice in the TA, pre, and pro groups did not decrease at the beginning of treatment (Figures 1(c) and 1(d)).In addition, the colons of the mice in the model group were significantly shortened, and the length of the colons in the TA group was increased compared to that in the model group (Figure S1).

Effect of TA on the Intestinal Barrier in Ulcerative Colitis.
The expression levels of claudin-1, occludin, and ZO-1 in the colons of mice in the model group were significantly lower than those in the colons of mice in the control group (P <0:05).The expression levels of claudin-1, occludin, and ZO-1 were significantly higher in the TA group than those in the model group (P <0:05).This suggests that intestinal barrier function is impaired in UC, and TA plays a role in regulating impaired intestinal barrier-related protein repair (Figure 2(a)-2(f)).In addition, MUC2 protein expression levels were decreased, and VEGF and TGF-β1 protein expression levels were increased in the colon of the model group.
Compared to the model group, MUC2 protein expression levels increased, and VEGF and TGF-β1 protein expression levels decreased in the TA group (Figure 3(a)-3(c)).

TA Alleviates Apoptosis in Ulcerative
Colitis.Compared to the control group, apoptotic cells were found in the damaged colon at this time point, and there were no significant numbers of apoptotic cells in the TA group compared to the model group (Figure 4(a)).Moreover, the amount of DAO was significantly higher in the model group (P <0:05) and lower in the TA group (P <0:05) than that in the control group (Figure 4(b)).Impaired colonic tissue and intestinal barrier function were associated with apoptosis of the colon cells.
3.4.TA Relieves Inflammation in Ulcerative Colitis.Apoptosis in the colonic tissue may be the reason for this.By detecting the levels of inflammatory signaling pathways and inflammatory factors in the colonic tissues, it was found that p-NF-kB inflammatory signaling pathway was activated in the model group.The expression levels of HMGB1, NLRP3, NF-kB, and p-NF-kB proteins were significantly lower in the TA group compared with the model group (P <0:05, Figure 5(a)-5(c)).Similarly, the levels of IL-10, IL-8, IL-17, and TNF-α were significantly higher in the model group than those in the control group (P <0:05), and the levels of IL-10, IL-8, IL-17, and TNF-α were significantly lower in the TA group than those in the model group (P <0:05; Figure 5(f )-5(i)).

TA Alleviates Oxidative Stress in Mice with Ulcerative
Colitis.Studies have shown that mice with UC exhibit severe inflammation, which leads to colonic tissue damage and triggering of apoptosis [40].The colonic barrier (outside of the central location in the GI tract) function is also impaired; therefore, we speculate that the location of the GI damage caused by UC may not always be the colonic site.The levels of oxidative stress and inflammatory factors in the serum of mice were found to be significantly lower in GSH and SOD (P <0:05) and higher in MDA, MPO, IFN-γ, FOXP3, IL-10, IL-8, IL-17, TNF-α, and RORγt (P <0:05) compared with the control group.Compared with the model group, the contents of GSH and SOD were significantly increased (P <0:05), and those of MDA, MPO, IFN-γ, FOXP3, IL-10, IL-8, IL-17, TNF-α, and RORγt were significantly decreased (P <0:05; Figure 6) in the TA group.As a result, oxidative stress is imbalanced, and inflammation spreads in the mice with UC.
3.6.TA Regulates the Intestinal Flora in Mice with Ulcerative Colitis.Interestingly, the Ang4 protein was found to be differentially expressed in the colonic tissues.Ang4 protein expression was significantly higher in the model group than that in the control group (P <0:05) and significantly lower in the TA group than that in the model group (P <0:05, Figure 7(a)).Activation of Ang4 phenotypes increases its bacteriostatic effect in the colon.Therefore, fecal samples and fresh intestinal contents from each group of mice were collected and analyzed for changes in the intestinal flora using 16S rDNA high-throughput sequencing.The sequence lengths of flora in feces and intestinal contents were concentrated at 404-431 bp, with the most abundant sequence length being 430 bp (Figure 7(b)).In the Greengenes database, the classify-sklearn algorithm of QIIME2 was used to obtain annotation information of all sequences into kingdoms, phyla, classes, orders, families, genera, and species.The resolution of annotations was higher in the model and TA groups than that in the control group.The specific composition of the microbial communities in the five groups at each taxonomic level was obtained by counting the ASV/OTU tables after drawing the level.It could be found that the number of taxonomic units was consistently highest in the model group except for the species class (Figure 7(c)).We conducted alpha and beta diversity analyses (Figures 7(e) and 7(f )) and found no significant differences in the Chao1 and Shannon indices between the groups.However, compared to the control group, the model group samples were well separated.Compared to the model group, the TA group achieved better separation.This result indicates that there was a significant difference in colony composition between the TA and model groups.Firmicutes and Bacteroidetes were the two most abundant phyla, accounting for over 80% of the total phyla level.Compared with the control group, the abundance of Firmicutes in the model group decreased, whereas the abundance of Bacteroides and Proteobacteria increased.Compared with the model group, the abundance of Firmicutes in the TA group increased, the abundance of Bacteroidetes decreased, and the abundance of Proteobacteria remained unchanged.This indicates that TA can effectively improve the imbalance of the gut microbiota in mice with colitis (Figure 7(g)).At the genus level, the control group had the highest relative abundance of Lactobacillus, which was  Mediators of Inflammation the dominant bacterium.Compared to the control group, the abundances of Lactobacillus, Alistipes, Coprococcus, Clostridium, Rikenella, and other bacteria in the model group were reduced, whereas the abundances of Bacteroides, Desulfovibrio, and other bacteria were increased.Compared to the model group, there was no significant change in Lactobacillus in the TA group, but there was an increase in Spirillum, whereas Bacteroides, Desulfovibrio Vibrio, and other bacteria were significantly reduced.In addition, the combined strain heat map revealed that Paraprevotella and Helicobacter were abnormally increased in the model group and decreased in the TA and pro groups (Figure 7(i)), which is similar to a previous analysis of the genus Chlorobi Phylum Oscillospira.

TA Modulates the Immune Response in Mice with
Ulcerative Colitis.In mice with PBMC, an increase in Th17 cells and a decrease in Treg cells were observed in the model group compared to those in the control group.UC induced the differentiation of monocytes into Th17 cells, and TA treatment reversed this trend (Figure 8).

TA Regulates Fatty Acid Homeostasis in Mice with
Ulcerative Colitis.Owing to the structural incompleteness of the mitochondria in the colon, damage to the digestive tract leads to changes in energy production, as can be seen by the changes in body weight of the mice, which appear to be reduced under conditions of a normal diet, suggesting to consider energy deficiency.We found that I-FABP protein was highly expressed in the model group and that TA reduced its expression (Figure 9(a)).SCFAs and acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acids were detected in the serum, and butyric acid was the most abundant (Figure 9(b)).Propionic acid was elevated in the model group compared to the control group (Figure 9(c)).Propionic acid levels were lower in the TA group than those in the model group (Figure 9(d)).
3.9.TA Regulates Bile Homeostasis in Mice with Ulcerative Colitis.As observed in the ileum, the expression levels of FGF15, ASBT, and BSEP proteins were significantly lower in the model group, whereas the expression levels of FXR  Mediators of Inflammation proteins were significantly higher in the control group (P <0:05).Moreover, the expression levels of FGF15, ASBT, and BSEP in the TA group were significantly higher than those in the model group, and the expression levels of FXR protein were significantly lower than those in the model group (P <0:05.Figure 10(a)-10(e)).We detected the metabolic profile of Bas in the serum by LC-MS and found that bile acid metabolism was abnormal in mice with UC; NorCA and GCA were found for the first time in the TA group compared to the control and model groups (Figures 10(f ) and 10(g)).Beta-MCA, NorDCA, and THDCA + TUDCA levels were elevated in the model group compared to those in the control group (Figure 10(h)).Compared to the model group, the beta-MCA, NorDCA, and THDCA + TUDCA levels were decreased in the TA group (Figure 10(i)).

Discussion
UC is an idiopathic inflammatory condition of the colon and the most common IBD worldwide.The pathology is characterized by increased fragility of the colonic wall, superficial erosion, and bleeding from the colonic wall.Inflammation occurs in the colonic mucosa and submucosa.DSS induces UC in mice.In the present study, lymphocyte infiltration, diffuse mucosal congestion and edema, a granular mucosal surface, increased mucosal fragility, and ulceration were observed in the colons of mice after DSS solution feeding.The expression level of intestinal barrier function proteins is reduced when the content of inflammatory factors in colonic cells increases, leading to the apoptosis of colonic cells.In addition, UC leads to increased levels of oxidative stress in mice, and abnormal levels of several factors have been observed in the serum.Alterations in the composition of the intestinal microbiota and mucosal immunodeficiency may contribute to UC [41].Therefore, we speculated that disruption of the intestinal barrier and the appearance of inflammation may be associated with alterations in the intestinal microbiota and immunodeficiency.Detection of DNA sequences in the intestinal contents and feces revealed a large number of different sequences between the model and control groups.Differences in Ang4 protein expression were also observed in the colon.Second, a Th17/Treg imbalance was found by detecting the Th17 and Treg immune cell levels.A large amount of Th17-produced IL-17 was present in the Mediators of Inflammation model group, and similarly, abnormally high expression of Foxp3 was observed.However, the imbalance of immune cells could not be the causative factor, and the dysbiosis of the intestinal flora was directly related to the colonic tissue.Therefore, the cause of these results might also start from the alteration of the intestinal flora.
Drugs for the treatment of UC are continuously being developed and utilized.In recent years, various novel therapeutic approaches using prebiotics, probiotics, commensal bacteria, and fecal microbial transplants as complementary and alternative medicines have improved the condition of patients with IBD [42].Probiotics can prevent human  Mediators of Inflammation intestinal diseases; modulate the host immune response and intestinal mucosa by acting on antigens and altering the composition of the intestinal microbiota [43][44][45]; and repair the damaged intestinal mucosal barrier in patients with UC during disease progression [46] by inhibiting HMGB1-mediated intestinal barrier dysfunction [47], modulating immunomodulatory activity to influence cytokine expression, and maintaining the intestinal permeability by reducing inflammation and protecting intestinal barrier integrity [48].Probiotics such as Bifidobacterium fragilis can improve the mucosal immune system in UC by interacting with the intestinal Treg and inducing IL-10 production [25].Mucin Muc2 is the major glycoprotein in the colonic mucus, and MUC2 deficiency can lead to impaired epithelial barrier function, intestinal flora imbalance, and spontaneous colitis, and studies have demonstrated that the probiotic mixture VSL#3 reduces colonic inflammation and improves intestinal barrier function in Muc2 mucin deficient mice [49].Thus, the intestinal flora can regulate intestinal barrier function, inflammation, and immune responses in mice with UC.TA regulates intestinal barrier function, inflammation, and the immune response [50][51][52].In this study, 16S   Mediators of Inflammation rDNA high-throughput sequencing was used to analyze the intestinal flora of mice.The results showed that the abundance of Firmicutes in the DSS-induced colitis mouse model was significantly reduced, and the abundance of Bacteroides and Denaturia was significantly increased.After TA intervention, the in the intestinal flora significantly improved, the abundance of Firmicutes significantly increased, and the abundance of Bacteroides significantly reduced.Similar to previous studies, kaempferol reshaped the gut microbiota by increasing the ratio of Firmicutes to Bacteroidetes [53].Zhang et al. [54] found that fecal microbiota transplantation increased the relative abundance of Firmicutes, reduced the abundance of Bacteroidetes, and restored the intestinal microbiota to normal levels.The study found that Shenlingbaizhusan increases the relative abundance of SCFA-producing bacteria at the genus level, including Oscillospira [55].In this study, the abundance of Osmospirillum spp. was increased after anisole intervention.Therefore, TA modulates intestinal flora in mice with UC and alleviates UC.By comparing the effects of TA and probiotics, we found that both TA and probiotics increased the expression levels of claudin-1, occludin, and ZO-1 proteins in the colon, but the effect in the TA group was stronger than that in the probiotic group.By comparing the results of HE and IHC staining, it was found that the TA group had a better effect on colonic mucosa repair than the probiotic group.In addition, the protein expression levels of inflammatory signaling pathways in the TA group were lower than those in the probiotic group compared to the model group.Finally, changes in immune cell content in the colonic group showed the same advantages.Ang4 protein expression in the colonic tissue was abnormally increased in the model group and significantly decreased in the TA group compared to that in the model group.Ang4 produced by mouse Paneth cells is secreted into the intestinal lumen and exhibits bactericidal activity against intestinal microorganisms induced by Bacillus mimicus [56].Thangamani et al. [57] found that taurocholic acid, a bile acid, inhibited the mRNA expression level of ang4 and regulated Candida albicans in the gastrointestinal tract.Combined with the LC-MS results, the percentage of secondary Bas was higher in the model group than that in the control and TA groups.This result suggests that  Mediators of Inflammation the increased flora in the colon of mice with UC is also involved in BA metabolism.Jia et al. [58] indicated that FGF15 plays an important role in energy metabolism, including BA homeostasis, glucose metabolism, and protein synthesis.FXR is a nuclear receptor expressed mainly in intestinal tissues that regulate BA, lipid, and glucose homeostasis [59].ASBT acts as a transporter protein responsible for ileal BA reabsorption [60].Impaired BSEP function may lead to cholestasis in humans [61].One study found that ASBT expression was reduced in murine, canine, and rabbit models of intestinal inflammation [62].Zhao et al. [63] found that a high-fat diet promotes DSS-induced UC by downregulating the FXR expression through the TGFB pathway.In the present study, we observed abnormally high FXR protein expression in mice with UC, and the protein expression levels of FGF15, ASBT, and BSEP were lower than those in the control group.After TA treatment, the expression levels of FXR protein were significantly reduced, and those of FGF15, ASBT, and BSEP were significantly increased; the increase was higher than that in the probiotic group.Laffin et al. [64] found that fatty acids modulate colitis susceptibility.Chen et al. [65] found that dandelion extract modulated fatty acid degradation and impaired microbial metabolism to ameliorate dextran sodium sulfate-induced colitis.SCFAs, anaerobic fermentation byproducts of undigested carbohydrates in the colon, were found to be an important energy source for the colonic epithelial cells [66].In the present study, the acetate, propionate, and butyrate levels were significantly lower in the model group than those in the control group.In addition, we found that I-FABP was abnormally highly expressed in the model group, suggesting that fatty acids are not properly metabolized in the colon of mice with UC, which leads to fatty acid aggregation.Fatty acids have inherent energy-supplying properties, and impaired fatty acid metabolism in the colon of mice with UC not only leads to a reduced energy supply to the organism but also affects the mucosal barrier function.In contrast, TA reduced the fatty acid content in the colon in this study, thus alleviating the subsequent chain reaction caused by abnormal fatty acid metabolism.

Mediators of Inflammation
In conclusion, this study demonstrated that TA can reshape the intestinal flora and reduce the production of fatty acids and BAs in DSS-induced UC and that the imbalance between inflammation and immunity in the colon of mice with UC is not only regulated by the intestinal flora but also by the reduced production of fatty acids and BAs.TA not only alleviated the impaired intestinal barrier function in mice with UC but also provided a superimposed effect on the restoration of colonic barrier function by reducing the synthesis of BAs; the same superimposed effect was also observed in Th17/Treg homeostasis.

Mediators of Inflammation
FIGURE 1: Continued.

FIGURE 1 :
FIGURE 1: DSS-induced colitis in BALB/c mice: (a) HE staining of the colon tissue, Scala bar = 50 μm; (b) ultrastructure of the colon was observed using transmission electron microscopy, Scala bar = 1 μm; (c) body weight changes in mice with UC; (d) body weight of mice with UC on day 15; (e) colon length of mice with UC; (f ) histological score.

FIGURE 7 :FIGURE 9 :FIGURE 10 :
FIGURE 7: Trans-anethole regulates the intestinal flora in mice with ulcerative colitis: (a) western blot detected the protein expression levels of Ang4; (b) the sequence lengths of flora in feces and intestinal contents; (c) distribution of the number of Taxa in each group; (d) sample twodimensional sorting chart for PCoA analysis; (e, f ) microbial diversity index of the microbial community; (g) phylum abundance; (h) genus abundance; (i) heatmap of taxa clustered.
Forty SPF, 6-8 weeks old, BALB/c mice weighing 20-25 g were obtained from Three Gorges University.All experiments were approved by the Animal Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd.(HLK-20220520-003) and performed in accordance with the National Institutes of Health Guidelines for the Care and Use of 2.1.Animal.All antibodies were purchased from Bioswamp (dilution, 1 : 200).