C57BL/6 mice (6–8 weeks of age, weighing 16–18 g) were acquired from the SPF Biotechnology Co., Ltd. (Beijing, China). Before the experiment, the mice were given 3 days to adapt to the laboratory environment. Throughout the adaptation and study periods, all animals were maintained on a 12-h light-dark cycle (23 ± 1°C with a relatively constant humidity of 50 ± 5%) under specific pathogen-free (SPF) conditions. The animal study was carried out according to the guidelines of the Animal Experimentation Ethics Committee, Tianjin University of Science and Technology.

All the mice were randomly divided into five groups, namely, the control group, model group, positive medicine group (SASP), low-dose apigenin (AP-L) group, and high-dose apigenin (AP-H) group (n = 6 per group). The mice in the control group drank water normally, and experimental mice were administered 2.0% (w/v) dextran sodium sulfate (DSS, MW, 36–50 kDa; Meilunbio, Dalian, China) in drinking water for 23 days to induce experimental colitis. After 2 days, mice in the model groups received 0.5% of CMC-Na, mice in the positive medicine group received 200 mg/kg of SASP, and mice in AP-L and AP-H groups received 150 and 250 mg/kg of apigenin, respectively, which was given by intragastric administration once a day for 21 days.

During the experiment, mice in each group were weighed every 2 days, and feces were collected to observe their traits. Fecal consistency, fecal occult blood, and weight loss were used for Disease Activity Index (DAI) scores. Mice were sacrificed under 0.5% pentobarbital sodium (50 mg/kg, I.P.) on day 26. Blood was centrifuged at 4,000 r/min at 4°C for 20 min, and serum was stored at −80°C for further analysis. The general morphology of colon samples was photographed. The length of the colon was measured. Colonic contents were collected and stored at −80°C. Colon tissues were fixed in 10% formalin for further analysis.

The colon samples were fixed, dehydrated, and embedded in paraffin. Tissues embedded in paraffin were cut into 3 μm sections. Before staining, the sections were deparaffinized and rehydrated. Hematoxylin-eosin (H&E) staining used standard techniques. Eosin (ZSGB-BIO, Beijing, China) was used to stain cytoplasm, and hematoxylin (ZSGB-BIO) was used to stain the nucleus. Alcian blue periodic acid-Schiff (AB-PAS) (LEAGENE, Beijing, China) staining was performed to measure the goblet cell quantity of colon tissue. The sections were stained with alcian blue for 15 min and oxidized with periodic acid for 5 min. Then, the sections were stained with Schiff dye for 15 min. All tissue sections were observed using an optical microscope (Olympus, Tokyo, Japan). Mucosal damage and inflammatory cell infiltration were used as study parameters to evaluate the histological scores.

Colon tissues were ground with physiological saline and centrifuged (3,000 rpm, 20 min) at 4°C. The supernatant was collected for further analysis. Cytokines including TNF-α, IL-6, IL-1β, and IL-10 in colonic tissues were measured by ELISA Kits (mlbio, Shanghai, China) according to the manufacturer’s instructions. Absorbance values were detected with a microplate reader. The cytokine concentration was obtained according to the standard curve.

The level of myeloperoxidase (MPO) in colonic tissues was measured by an MPO activity assay kit (mlbio, Shanghai, China). The detection method was carried out according to the manufacturer’s instructions, and the absorbance value was measured at 460 nm.

Colon tissues were washed with phosphate-buffered saline (PBS) and lysed fully in ice-cold lysis buffer with the Protease Inhibitor Cocktail (MCE, New Jersey, USA) on ice. Lysates were separated by electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). The membranes were blocked with 5% skim milk powder and incubated with primary antibody against ZO-1 (1:1,000, Bioss), Occludin (1:500, Bioss, Beijing, China), Claudin1 (1:1,000, Bioss), and GAPDH (1:25,000, Proteintech, Chicago, USA) at 4°C overnight and then incubated with a horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody (1:5,000, YEASEN, Shanghai, China) for 1 h at room temperature. The intensity of protein was assessed using a chemiluminescent imaging system (Image Quant LAS 4000, Atlanta, USA).

Immunohistochemistry (IHC) was conducted according to the previously former method (15). Colon tissue embedded in paraffin was sliced (3 μm) and then deparaffinized and rehydrated with dimethyl-benzene and alcohol. Next, samples were incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity and then blocked with goat serum (Proteintech, Chicago, USA) for 1 h. After being blocked, the samples were incubated with the following primary antibodies overnight at 4°C: ZO-1 (1:250, Bioss, Beijing, China), Occludin (1:400, Bioss), and Claudin1 (1:200, Bioss). The tissues were washed three times with PBS for 5 min each time. After that, the primary antibody in the negative group was displaced by PBS. The secondary antibody was subsequently added with the horseradish peroxidase-polymer anti-mouse/rabbit IHC kit (Maixin Biotech, Fuzhou, China) for 1 h at room temperature. 3,3′-Diaminobenzidine-tetrahydrochloride (DAB) was used to stain the tissues and counterstained with hematoxylin. All the samples were observed with a microscope (Olympus, Tokyo, Japan). The immunohistochemistry score was calculated by multiplying the intensity (0 = negative, 1 = canary yellow, 2 = claybank, 3 = brown) and the positive cell percentage scores (1 = less than 25%, 2 = 25–50%, 3 = 51–75%, 4 = more than 75%).

The short-chain fatty acids in the colonic contents were measured by GC-MS (SHIMADZU, GCMS-QP2010 Ultral, Kyoto, Japan). The method of SCFA determination is according to a previously described method (16). After the fecal samples were dissolved and homogenized, the mixture of sulfuric acid and ether was added to extract SCFAs and then centrifuged at 4°C, 12,000 rpm for 15 min to prepare the samples. Then, anhydrous sodium sulfate was added to the supernatant and centrifuged at 4°C (12,000 rpm, 15 min). The supernatant was collected and injected into GC-MS. The standard SCFAs are a mixture of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid (MACKLIN, Shanghai, China).

Genomic DNA was extracted from colonic contents. DNA pyrosequencing of the 16S rRNA V3-V4 region was completed in Novogene Co., Ltd. (Beijing, China). The primers 341F and 806R (forward 5′-CCTAYGGGRBGCASCAG-3′ and reward 5′-GGACTACNNGGGTATCTAAT-3′) were used to amplify DNA. Bioinformatics analysis was performed on the Illumina MiSeq platform of Novogene.

All data are presented as means ± SD. ANOVA test (one-way or two-way, with post-hoc tests) was performed using GraphPad Prism 7.0 software (^*P ≤ 0.05, ^**P ≤ 0.01).

To investigate whether apigenin has a therapeutic effect on UC, mice were treated with 2.0% DSS in drinking water, while apigenin was administered by gavage from day 3 to day 24 (Figure 1A). As expected, DSS markedly reduced the body weight compared to normal mice. In contrast, SASP significantly increased body weights, and apigenin ameliorated weight loss in a dose-dependent manner in mice induced by DSS (Figure 1B) (P < 0.01). DAI score based on stool consistency, bloody stool, and weight loss assessments were also evaluated. DSS could significantly increase the DAI, whereas SASP and apigenin treatment could improve this phenomenon (Figure 1C) (P < 0.01). In addition, it was reported that DSS-induced colitis developed symptoms of spleen hypertrophy (17). The results showed that apigenin inhibited spleen hypertrophy in colitis mice compared to the mice in the DSS-induced colitis group (Figure 1D) (P < 0.01). Overall, these results indicated that apigenin administration treatment significantly ameliorated DSS-induced UC.

To further investigate the therapeutic effect of apigenin on UC intuitively, we compared the length, gross morphology, and tissue damage of colorectal sites in each group of mice. DSS typically causes colonic shortening while such change was also improved by 150 and 250 mg/kg of apigenin. Moreover, there was no significant difference in colorectal length between high-dose apigenin-treated mice and SASP-treated mice (Figure 2A) (P < 0.05). The colorectal tissue displayed obvious fold, good elasticity, and reflectance in the wild mice. Colon tissues from UC mice displayed indistinguishable upper colorectal folds, granular mucous membranes, multiple hyperemia and swelling, and surface ulcers. Apigenin ameliorated these colonic damages and reduced colon macroscopic damage scores (Figure 2B) (P < 0.01). Histological examination of the hematoxylin and eosin-stained sections of the colon revealed severe inflammatory lesions, crypt erosion, and inflammatory cell infiltration from UC mice. Strikingly, SASP and apigenin-treated mice exhibited less inflammatory cell infiltration and intact colonic architecture without mucosal damage, reducing microscopic damage scores (Figure 2C) (P < 0.05). AB-PAS staining results demonstrated that compared with the control group, there was a lower goblet cell quantity of colon in model mice, while apigenin administration restored the damage induced by DSS (Figure 2D) (P < 0.01). Overall, apigenin supplementation could effectively alleviate the severity of colon injury in mice with UC.

To demonstrate the effect of apigenin on inflammation, inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) were determined. Increased proinflammatory cytokines TNF-α, IL-1β, and IL-6 expressions were observed in the model group. Compared with mice induced by DSS, SASP and apigenin treatment decreased TNF-α, IL-6, and IL-1β levels (Figure 3A) (P < 0.05). IL-10 produced by intestinal CD₄⁺ T cells is responsible for maintaining immune homeostasis, thereby inhibiting colitis development (18). The results showed that apigenin increased the level of IL-10 in the colitis of DSS-induced colitis mice (Figure 3B) (P < 0.05). MPO, an enzyme present in neutrophils, is positively correlated with the concentration of neutrophils in the inflammatory region, and increased MPO activity is an indicator of neutrophil infiltration and inflammation (19). In the model group, MPO activity increased, and apigenin treatment inhibited neutrophil infiltration and inflammation in UC mice (Figure 3C) (P < 0.01). It is noteworthy that apigenin had an effect on TNF-α, IL-6, IL-1β, IL-10, and MPO in a dose-dependent manner. These results suggested that apigenin alleviated inflammation and kept intestinal homeostasis.

Gut barrier damage was the moment pathological characteristic of colitis (2), such as UC. The zonula occluden (ZO), occludin, and claudin family proteins are essential tight junction proteins of epithelial barrier (20). The levels of tight junction proteins were determined through Western blot analysis and IHC analysis. As shown in Figure 4A, the protein expressions of ZO-1, claudin-1, and occludin in the model group were reduced, while the supplement of SASP and apigenin could improve the expression of these proteins. Furthermore, the IHC images and staining scores indicated that substantial loss of staining intensity of ZO-1, claudin-1, and occludin in mice induced by DSS was observed compared with the control group (Figures 4B, C) (P < 0.01). However, SASP and apigenin might improve this change, and high-dose apigenin might have a better effect compared with SASP. The above results demonstrated that apigenin prevented gut barrier damage in DSS-induced colitis mice.

As metabolites of intestinal microflora, SCFAs maintain intestinal function by inhibiting inflammation and protecting the intestinal epithelial barrier (21). We detected the contents of SCFAs using GC-MS. Compared with the control group, the concentration of SCFAs in model groups was downregulated (P < 0.01). But apigenin and SASP administration significantly increased the level of SCFAs, and the SCFA content of AP groups was almost as effective as positive drugs (Figure 5) (P < 0.05).

To investigate whether apigenin has a moderating effect on the composition and diversity of gut microbiota, we performed 16S rRNA gene sequencing analysis of the colonic content of different groups of mice. Alpha diversity, such as Chao1 and ACE index, was recovered in apigenin and SASP groups (Figure 6A). To explore the specific variations in different groups, we evaluated the relative abundance of taxonomic groups. At the phylum level, we found that the dominant phylum was Firmicutes, Verrucomicrobia, and Proteobacteria. The results showed that DSS induction contributed to decreases in Firmicutes and Verrucomicrobia and increases in Proteobacteria. Apigenin and SASP intervention could significantly reverse the DSS-induced variation in the relative abundance of Verrucomicrobia and Proteobacteria (Figure 6B).

In the top 35 genus level, 16 genera were downregulated and 12 genera were upregulated in the DSS-induced mice, while SASP and apigenin supplementation selectivity reversed this change (Figure 6C). Among them, the relative abundances of Lactobacillus, Dubosiella, Akkermansia, and Faecalibaculum were decreased and Turicibacter, Klebsiella, Romboutsia, and Bacteroides were increased at genus level in DSS-induced mice. Apigenin and SASPs administration selectively reversed the relative abundance (Figure 6D). Previous studies proved that Lactobacillus (22, 23), Dubosiella (24), Akkermansia (25, 26), and Faecalibaculum (27) are probiotics protecting the intestinal immune barrier from damage. Turicibacter (28, 29), Klebsiella (30), Romboutsia (31), and Bacteroides, pernicious bacteria in IBD, might have a positive correlation with the malignant development of UC. In addition, linear discriminant analysis (LDA) showed the main feature of the dominant flora among five different groups (Figure 6E). Our results clarified that DSS treatment altered species diversity of intestinal microbial homeostasis, while apigenin restored that to the normal level.

To investigate the effect of apigenin supplementation on the potential metabolic pathways of gut microbiota in DSS-induced mice, Tax4Fun analysis was executed. As shown in Figure 6F, apigenin notably reversed the changes in many basic metabolic pathways such as carbohydrate metabolism and amino acid metabolism. Furthermore, we analyzed the metabolic changes of gut microbiota related to immune diseases, cell growth, and cell death. The results indicated that apigenin possibly had a close correlation in ameliorating the primary immunodeficiency, systemic lupus erythematosus, apoptosis, and tight junction motivated by DSS (Figure 6G). These findings demonstrated that treatment of apigenin may be effective in restoring dysregulated gut microbiota, which might play its role in the metabolite of bacteria. The protective mechanism of apigenin against UC is shown in Figure 7.