All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Guangdong Medical University and the experiments were approved by the Ethics Committee of Shunde Women and Children's Hospital of Guangdong Medical University (approval no. 2023054; Foshan, China).

An enriched environment, nutritious diet and humane handling were provided to the animals. All the mice were provided by the Guangdong Medical Laboratory Animal Center and housed in a specific pathogen-free facility (temperature, 23±1˚C; 12/12 h light/dark cycle; humidity, 50-60%). A single mouse was used as the experimental unit and the placement of cages was random throughout the space used. Measures were taken to ensure the welfare of the animals and to minimize discomfort for all animals involved in the present study. Animals were fed daily with a fresh diet to maintain body weight and normal growth. All mice were allowed ad libitum access to food and water. The criteria used to determine when animals should be euthanized included, but were not limited to, loss of body weight of 20% compared with the body weight before the study began, severe behavioral abnormalities or physiological distress. In the present study, a maximum weight loss of 19.78% was observed. The mice were euthanized by intraperitoneal injection of 100 mg/kg pentobarbital sodium. Death was verified by checking for the cessation of the heartbeat and pupil dilation.

The experiments were performed during the 12 h light cycle. For RNA-sequencing (RNA-seq) experiment, a total of 6 healthy male C57BL/6 mice (body weight, 18-22 g; age, 8 weeks) were randomly divided into two groups (n=3/group). The control group received distilled water, while the treatment group received 3.5% DSS for 7 days. For the intervention study, a total of 18 healthy male C57BL/6 mice (body weight, 18-22 g; age, 8 weeks) were randomly divided into three groups (n=6/group): i) Control group; ii) DSS group (3.5% DSS) and iii) DSS + XAV939 group [DSS + XAV, 3.5% DSS + 10 mg/kg body weight (BW) XAV939]. In our previous study, we did not observe colitis-related changes when utilizing low concentrations of DSS to induce the UC model (unpublished data). Subsequently, findings from other literature were cross-referenced and the experimental model of DSS administration was adjusted accordingly (20,21). Throughout the experiment, there were no instances of animal mortality observed prior to its conclusion. The dose of XAV939 was determined in accordance with the report conducted by Distler et al (22). DSS was dissolved in drinking water and XAV939 was dissolved in DMSO (≥99.7%; cat. no. D2650; Sigma-Aldrich, Inc.) and 0.4 ml was administered by intraperitoneal injection for 7 days. The control and DSS groups were injected with 0.4 ml PBS mixed with the same concentration of DMSO (Fig. S1). All mice were euthanized and the entire colon was collected. The length of colons, average daily weight gain and weight of the colon per unit length were examined to measure the treatment outcome. DSS was purchased from Dalian Meilun Biology Technology Co., Ltd. and XAV939 was purchased from MedChemExpress (cat. no. HY-15147).

RNA extraction and purification from colonic crypts were carried out in accordance with the manufacturer's instructions (cat. no. 74104; Qiagen GmbH). The total RNA of colonic crypts from the control and DSS groups were sequenced by Guangdong Magigene Biotechnology Co., Ltd. The Agilent 4200 Bioanalyzer (Agilent Technologies, Inc.) was used for quality control of samples. All samples had RNA integrity numbers >8. Subsequently, 150 bp paired-end reads were generated using the Illumina NextSeq sequencing platform (Illumina, Inc.).

Raw reads were first trimmed with Trimmomatic (version 0.36; http://www.usadellab.org/cms/index.php?page=trimmomatic) to acquire the clean reads, which were then mapped to National Center for Biotechnology Information Rfam databases (version 14.10; https://rfam.org) and the rRNA sequences removed using Bowtie2 (version 2.33; https://github.com/BenLangmead/bowtie2). The reads were mapped to the mouse reference genome (Mus_musculus_GCF_000001635.27) using Hisat2 (version 2.1.0; https://github.com/infphilo/hisat2) (23,24). HTSeq-count (version 0.9.1; http://htseq.readthedocs.io/en/release_0.9.1/) was used to obtain the read count and function information of each gene. The count tables were normalized based on their library size using trimmed mean of M-values normalization implemented in R/Bioconductor EdgeR (version 3.34.0; http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) (25,26). Normalized read counts were fitted to a negative binomial distribution with a quasi-likelihood F-test. Principal component analysis (PCA; https://www.r-project.org) was performed for the regularized log transform of the normalized counts using plotPCA tools with default parameters (27,28). Differential gene expression analysis was further carried out using EdgeR. The log2 fold change <-1 or >1 and a P-value <0.05 were used to examine differentially expressed genes (DEGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis was also used to calculate DEG enrichment, which could be used to obtain information on the fold changes of expressed genes at the molecular level. To determine the metabolic and signaling pathways, the DEGs at various KEGG pathway levels were counted. The log2 fold change <-1 or >1 and a P-value <0.05 were used to examine differentially for KEGG and GO analyses.

The colons were collected and fixed in 4% paraformaldehyde, with fixation typically conducted at room temperature (20-25˚C) for 24 h. Following fixation, the colons were paraffin embedded and sectioned at a thickness of 5 µm for histological analysis and IHC. Hematoxylin and eosin (H&E) staining was used to assess morphological changes in the intestine, and was imaged (Evos XL Core; Thermo Fisher Scientific, Inc.). Tissue sections were dewaxed and rehydrated, and then stained with hematoxylin for 5 min to highlight nuclear structures. Next, the sections were rinsed and counterstained with eosin for 1 min to color the cytoplasm and extracellular matrix. The staining was carried out at room temperature (20-25˚C). Finally, the sections were dehydrated, cleared and mounted onto slides for microscopic examination. Antigen repair was performed on paraffin sections using EDTA antigen repair solution (cat. no. ZLI-9067; ZSBG-BIO) in boiling water for 10 min, then blocked with fetal bovine serum at room temperature for 1 h. IHC staining for IL-1β (cat. no. ab234437; Abcam), SOX9 (cat. no. ab185230; Abcam), β-catenin (cat. no. R22820; Zenbio), Villin (cat. no. SC58897; Santa Cruz Biotechnology, Inc.) and PPAR-γ (cat. no. 16643-1-AP; Wuhan Sanying Biotechnology) was performed. The dilution ratio of primary antibody was 1:100, and the incubations for primary antibodies were conducted at 4˚C for a duration of 16 h. The samples were then washed with PBS three times and incubated with Cy3 (cat. no. K1209; APeXBIO Technology LLC) and FITC (cat. no. IF-0091; Beijing Dingguo Changsheng Biotechnology Co., Ltd.)-labeled fluorescent secondary antibodies (1:500) for 1 h at room temperature. The dilutions for all antibodies used were prepared using a universal antibody dilution buffer (cat. no. WB500D; New Cell & Molecular Biotech Co., Ltd.). The nuclei were stained with DAPI (cat. no. C55-141215; Biosharp Life Sciences) (1:1,000 dilution) for 10 min at room temperature. Images were obtained using a confocal microscope (LSM 900; Carl Zeiss AG). The fluorescence signal intensity was measured with ImageJ software (ImageJ2x 2.1.4.7; National Institutes of Health), and the minimum value in the control group was set to 1.

The RNA was extracted from cultured intestinal organoids. RNA extraction was performed was performed using a FastPure cell/tissue RNA isolation kit V2 (cat. no. RC112-01; Vazyme Biotech Co., Ltd.), according to the manufacturer's instructions. The reverse transcription process followed RNA extraction using a reverse transcription kit (HiScript II Q RT SuperMix for qPCR; cat. no. R222-01; Vazyme Biotech Co., Ltd.), which involved incubating the reaction mixture at 50˚C for 15 min and subsequently at 85˚C for 30 sec. The primers were purchased from Sangon Biotech Co., Ltd. (Table I). qPCR of TNF-α, IL-1β, Villin, peroxisome proliferator-activated receptor γ (PPAR-γ), SOX9 and β-catenin was performed. To normalize gene expression, a GAPDH endogenous control was used. The ChamQ Universal SYBR qPCR Master Mix (cat. no. Q711-02; Vazyme Biotech Co., Ltd.) and qPCR was employed to verify the mRNA expression using a platform from Applied Biosystems; Thermo Fisher Scientific, Inc. The reaction conditions set were as follows: An initial denaturation at 94˚C for 5 min; followed by 36 cycles of (denaturation at 94˚C for 30 sec, annealing at 60˚C for 30 sec, and extension at 72˚C for 30 sec); a final extension at 72˚C for 5 min; and a subsequent incubation at 60˚C for 30 sec followed by 95˚C for 30 sec. PCR amplification was analyzed using the comparative 2^-ΔΔCq method (29).

The crypts were isolated by taking ~5 centimeters of colon and splitting it longitudinally. The intestinal contents were then cleaned using PBS and transferred to a 15 ml centrifuge tube and 5 ml of PBS was added. After gently shaking by hand for 5 min, the solution was replaced with fresh PBS and the shaking process was repeated six times. The colon segments were then cut into 1 cm pieces. The PBS was discarded, and a solution containing 30 mmol/l EDTA-2Na and 1.5 mmol/l dithiothreitol in PBS was added. The solution was gently shaken by hand for 5 min, then the separation solution was replaced and the shaking process was repeated once. Fresh PBS was added, and the suspension was observed for crypt units and debris impurities. When there were more crypt units and less debris impurities, fresh PBS was added and the sample was shaken vigorously by hand for 1 min, incubated on ice for 5 min and the crypts were collected. The quality of the isolated crypts served a crucial role in the success of organoid culture. The crypts obtained exhibited intact structures and a high purity (>90% of them retained the intact structure of the crypt base), ensuring the quality of the resulting organoids (Fig. S2). Crypts were embedded in Matrigel and cultured using a mouse colonic organoid kit (cat. no. K2204-MC; Biogenous), and the seeding density of the crypts was 5/1 µl of matrix gel, with 8 µl per well (40 crypts in total). The organoid was cultured in a CO₂ incubator at 37˚C. At 24 h post-crypt seeding, a gradual transformation of the crypts into rounded and transparent structures was observed using a Evos XL Core microscope (Thermo Fisher Scientific, Inc.), which is a characteristic feature of organoid formation. The intestinal organoid was induced with TNF-α to establish an inflammatory model. TNF-α and XAV939 treatment was performed after 48 h of seeding, and the sample in each well served as a replicate and was collected after 24 h of treatment. The intestinal organoids were divided into three groups: i) Control group; ii) TNF-α group treated with 100 ng/ml TNF-α (cat. no. 10602-R101-F; Sino Biological, Inc.); and iii) TNF-α+XAV939 group treated with 100 ng/ml TNF-α and 10 µM XAV939. The dose of XAV939 used was determined in accordance with the previous report by Liang et al (30). The organoid formation efficiency was calculated by dividing the number of colonies formed by the number of crypts seeded and expressing this as a percentage. The surface area was measured using ImageJ software (ImageJ2X 2.1.4.7, National Institutes of Health).

The colon samples collected from various groups in the XAV939 experiment were used to prepare protein samples. The colon was lysed by RIPA (cat. no. WB3100; New Cell & Molecular Biotech Co., Ltd.) and prepared into protein samples of the same concentration by the BCA kit (cat. no. P0011; Beyotime Institute of Biotechnology). The sample loading amount per lane was 10 µg. Protein samples were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin at room temperature for 1 h. The membranes were incubated with primary antibodies at 4˚C for 12-16 h. The selection and use of these primary antibodies were consistent with the description for IHC: IL-1β (cat. no. ab234437; Abcam), SOX9 (cat. no. ab185230; Abcam), β-catenin (cat. no. R22820; Zenbio), Villin (cat. no. SC58897; Santa Cruz Biotechnology, Inc.) and PPAR-γ (cat. no. 16643-1-AP; Wuhan Sanying Biotechnology). The dilution ratio of primary antibody was 1:1,000, and the dilutions for all antibodies used were prepared using a universal antibody dilution buffer (cat. no. WB500D; New Cell & Molecular Biotech Co., Ltd.). Samples were washed using TBS with Tween-20 (0.1%) three times and incubated with a HRP-goat anti-rabbit secondary antibody (cat. no. RGAR001; Wuhan Sanying Biotechnology) and a HRP-goat anti-mouse secondary antibody (cat. no. SA00001-1; Wuhan Sanying Biotechnology) for 1 h at room temperature. The enhanced chemiluminescence (KF8001; Affinity Biosciences) signals were scanned using a ChemiDOC XRS+ (Bio-Rad Laboratories, Inc.), and the band densities were analyzed using ImageJ software.

GraphPad Prism (version 8.0; Dotmatics) software was utilized for statistical analysis. All data were presented as the mean ± SEM. Multiple comparisons were performed using a one-way ANOVA and Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The colonic crypts were isolated for RNA-seq (Fig. 1A). PCA results showed a significant difference between the control group compared with the DSS group (Fig. 1B). A total of 212 genes were upregulated and 315 genes were downregulated after DSS treatment (Fig. 1C). KEGG analysis demonstrated that DSS treatment resulted in the differentially expressed genes related to various types of metabolic processes (Fig. 1D). Additionally, KEGG analysis showed significant changes in inflammatory cytokine receptor interactions and MAPK signaling pathways (Fig. 1E). The DSS group showed significant down-regulation of intestinal goblet cell and enterocyte marker genes, and a significant increase in secreting cell progenitor marker genes, which indicated a potential disorder in ISC differentiation (Fig. 1F). Furthermore, DSS significantly altered the gene abundance of inflammatory factors and other niche factors, which is crucial for the microenvironmental homeostasis of ISCs (Fig. 1F).

To confirm the therapeutic effect of XAV939 on DSS-induced UC in mice, the length of the colon was measured (Fig. 2A). Compared with the control group, DSS caused significant shortening of the colon (Fig. 2B, P<0.01) and a significant reduction in average daily weight gain (Fig. 2C, P<0.01). However, the results showed that treatment with XAV939 had no significant effect on the colon length compared with the DSS group (Fig. 2B, P=0.95). Additionally, XAV939 did not significantly improve the average daily weight gain compared with the DSS group (Fig. 2C, P=0.98). XAV939 treatment caused an increase in the weight of the colon per unit length compared with the DSS group (Fig. 2D, P=0.09), which indicated that the colon injury treated by DSS was not improved. Therefore, XAV939 may not be beneficial in mitigating the colonic changes associated with DSS-induced UC in mice.

The results of H&E staining showed that XAV939 treatment caused no significant difference in colonic epithelial structure and inflammatory infiltration compared with the DSS group (Fig. 3A). The increase in crypt depth, which can be considered a marker of intestinal damage, was significantly more pronounced after XAV939 treatment compared with the DSS group, which indicated that there was improvement in colon injury (Fig. 3B, P<0.01). Furthermore, the results of western blotting demonstrated that, compared with the control group, the DSS group exhibited a significant increase in IL-1β protein expression levels (Fig. 3C and D, P<0.05). The non-specific bands in the GAPDH blot may have been due to the high concentration of antibodies used. The results of IHC also demonstrated that the DSS induced a significant increase in the expression level of IL-1β (Fig. 3E and F, P<0.05). Notably, supplementation with XAV939 did not lead to significant changes in IL-1β protein expression levels (Fig. 3D, P=0.38; Fig. 3F, P=0.09). These findings collectively suggested that XAV939 had no beneficial effect on DSS-induced colon injury and inflammation.

Due to the severe damage caused by DSS to the intestinal epithelium and the key role of ISCs in promoting the repair process of the epithelium, the intestinal organoid model was used to verify the anti-inflammatory effect of XAV939 (Fig. 4A). These results demonstrated that XAV939 significantly reduced the forming efficiency of organoids compared with the TNF-α group (Fig. 4B, P<0.01), and significantly alleviated the TNF-α-induced increase in organoid area (Fig. 4C, P<0.01). TNF-α did not affect the forming efficiency of the organoid, but the stimulation was successful because TNF-α treatment led to a significant increase in TNF-α (Fig. 4D, P<0.01) expression level, and an increase in IL-1β (Fig. 4E, P=0.81) expression level. Nevertheless, the addition of XAV939 did not significantly reduce the expression levels of IL-1β and TNF-α induced by TNF-α treatment (Fig. 4D, P=0.47). Notably, the addition of XAV939 contributed to an increase in IL-1β expression levels (Fig. 4E, P<0.01), which indicated that XAV939 did not significantly relieve inflammation.

As the differentiation of ISCs is regulated by the Wnt/β-catenin signaling pathway, the protein expression levels of β-catenin and its downstream target SOX9 were examined (Fig. 5A). DSS significantly increased β-catenin (Fig. 5B, P<0.01) and SOX9 (Fig. 5B, P<0.05) protein expression levels, while XAV939 significantly reduced the protein expression levels of β-catenin (Fig. 5B, P<0.05) and SOX9 (Fig. 5B, P<0.01) compared with the DSS group. Simultaneously, β-catenin and SOX9 protein expression was determined by IHC (Fig. 5C). DSS significantly increased the fluorescence intensity of β-catenin (Fig. 5D, P<0.05) and SOX9 (Fig. 5D, P<0.01), while XAV939 significantly reduced the fluorescence intensity of β-catenin (Fig. 5D, P<0.05) and SOX9 (Fig. 5D, P<0.01). In the intestinal organoid experiment, XAV939 treatment significantly reduced the TNF-α-induced increase in the expression levels of β-catenin (Fig. 5E, P<0.05) and SOX9 (Fig. 5E, P<0.01). These results indicated that XAV939 could reverse the excessive activation of Wnt/β-catenin induced by DSS and TNF-α.

Previous studies have suggested that the PPAR-γ signaling pathway serves a role in enterocyte differentiation, and Villin serves as a marker for enterocytes in the intestine (31,32). To further investigate the regulatory effect of XAV939 on enterocyte differentiation, western blotting was used to assess the protein expression levels of PPAR-γ and Villin (Fig. 6A). The results demonstrated that DSS treatment led to decreased protein expression levels of PPAR-γ (Fig. 6B, P<0.01) and Villin (Fig. 6B, P<0.05). However, XAV939 did not significantly reverse this decreased expression (Fig. 6B). Simultaneously, PPAR-γ and Villin protein expression was determined by IHC (Fig. 6C). DSS significantly decreased the fluorescence intensity of PPAR-γ (Fig. 6D, P<0.01) and Villin (Fig. 6D, P<0.05), while XAV939 did not significantly reverse the reduction of the fluorescence intensity of PPAR-γ (Fig. 6D, P=0.31) and Villin (Fig. 6D, P=0.99). In the intestinal organoid model, XAV939 significantly reduced the expression levels of PPAR-γ (Fig. 6E, P<0.01) and Villin (Fig. 6E, P<0.01) compared with the TNF-α group, which indicated that the organoid was more sensitive to the inhibitory effects of XAV939 compared with the ISC in vivo.