Chemicals and antibodies
Reagents and Kits: The bergenin (purity 98.6%) used in this study was purchased from Sigma–Aldrich (St. Louis, MO, USA).
Antibodies: Primary antibodies targeting TLR4, 5’-BrdU, α-SMA, and iNOS were obtained from Cell Signaling Technology (Danvers, AP, USA). FITC-conjugated secondary antibodies against mice IgG and rabbit IgG were purchased from Signaling Technology (Danvers, AP, USA). All antibodies were utilized following the recommended dilutions.
Animals
Female KM mice of 10 weeks of age, with Specific Pathogen Free (SPF) grade, were obtained from the SLAC Company Limited (Pujiang, Shanghai, China). The mice were housed in groups of three per cage under controlled conditions of 25°C and a 12-hour light/12-hour dark cycle. All experiments performed on the mice were approved by the Animal Ethics Committee of the Chinese Academy of Sciences and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (certificate number: NWIPB-2018–63).
After one week of acclimatization, the mice were randomly divided into three experimental groups (n = 9) and fed a germ-free diet of SPF grade. This diet consisted of 100 g of water, 220 g of protein, 40 g of fat, 50 g of crude fiber, 15 g of Calcium, 10 g of P, 12 g of lysine, and 7 g of methionine and cysteine per kilogram obtained from Xietong Biotechnology Co. Ltd., (Jiangsu, China) (certificate number (2014) 01008). The drinking water (18 MΩ•cm–1, germ-free, and high purity) and corncob bedding were also germ-free. The mouse cages were cleaned and sterilized daily under ultraviolet lamps, and the water feeders were sterilized daily using high-pressure steam (121°C, 20 mins). The study consisted of three groups (n = 9): (1) Control, which received an SPF-grade diet without DSS treatment; (2) DSS, which received an SPF-grade diet with DSS treatment; and (3) DSS + BE, which received an SPF grade diet with DSS and bergenin treatment through intragastric route, at a dose of 50 mg/Kg/d. The dose of BE was determined based on previous literature 28 and our prior study.
After one week of receiving SPF diets, the mice in the DSS and DSS + BE groups received DSS (molecular weight 36–50 kDa; Sigma–Aldrich, St. Louis, MO, USA) in their drinking water at a concentration of 2.5% for seven consecutive days. From day 8 to day 10, the drinking water was switched to standard high-purity germ-free water (18 MΩ•cm–1). Fresh DSS solution was prepared daily and filtered through a 0.22 µm filter membrane. The control group mice received standard high-purity germ-free water (18 MΩ•cm–1) throughout the study. Daily records were maintained for food and water consumption.
Evaluation of pathological phenomenon
Body weight, stool consistency, and rectal bleeding of the mice were monitored and recorded daily. Stool consistency and rectal bleeding were evaluated and scored based on the established methods. Stool consistency scores ranged from 0 (normal), 1 (soft stools), 2 (very soft stools), and 3 (watery stools), while rectal bleeding scores ranged from 0 (negative hemoccult), 1 (positive hemoccult), 2 (trace amounts of blood), and 3 (visible rectal bleeding).
Tissue Collection
On the tenth day, the mice were anesthetized with chloral hydrate via intraperitoneal injection. Animals were then humanely euthanized by cervical dislocation, and their abdominal cavities were opened to remove the large intestine, which was placed on an ice plate. The length of the colon, measured from the colorectal junction to the anus, was recorded. Additionally, the spleen index, kidney index, and liver index were also recorded.
Histopathological Analysis
The colon tissues were fixed in 10% formalin for 48 h and processed for frozen sectioning. 3 µm thick sections of the colon were stained with hematoxylin and eosin (H&E). Eight randomly selected fields were viewed under a light microscope, and each field was given scores based on the inflammation severity (ranging from 0–3), inflammation extent (ranging from 0–3), and crypt damage (ranging from 0–4). The total histologic scores for each mouse were calculated by summing the scores from all three parameters, with a maximum possible overall score of 10. A detailed description of the standard scores used is provided in the supplementary materials (Table S1).
Immunofluorescence Microscopy
Colon sections were cultured on glass coverslips and fixed with 4% paraformaldehyde at room temperature for 15 min. The sections were then permeabilized using 0.1% Triton X-100 in PBS for 5 min. Non-specific antibody binding was blocked by incubating the sections with 3% BSA-PBS for 30 min. The primary antibody (β-catenin) was diluted in 0.2% BSA-PBS and incubated with the sections for 2 h at Room temperature. After thorough washing, an Alexa Fluor-488 conjugated secondary antibody (Thermo Scientific, Waltham, AP, USA) was added to bind the primary antibodies. Hoechst 33258 dye (Thermo Scientific, Waltham, AP, USA) was used to stain the nuclei, and the samples were mounted in ProLong Diamond anti-fade medium (Thermo Scientific, Waltham, AP, USA). Images were captured using a Nikon C1 fluorescence microscope and analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).
Analysis of stool microbiota composition by 16S ribosomal RNA gene sequencing
Each group of animals were housed in three cages, with three animals per cage. The cages were cleaned daily, and on day 10, stool samples were collected from each cage, snap-frozen in liquid nitrogen, and stored at − 80°C until further analysis.
Total bacterial DNA was isolated from frozen stools using a QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA). The V3-V4 region of the 16S rRNA gene was amplified, and DNA libraries were constructed following previously described methods. Paired-end sequencing, with a read length of 2 × 250 bp, was performed on the Illumina MiSeq platform (Illumina, Inc, San Diego, CA, USA). After demultiplexing, paired-end reads were joined using FLASH v1.2.7, followed by quality filtering and removal of chimeras. The resulting tags were assigned to operational taxonomic units (OTUs), with a 97% pairwise identity threshold, using UCLUST (version 1.2.22). The assigned taxonomy was aligned against the Silva reference database (Release128, http://www.arb-silva.de). For α-diversity analysis, Mothur (version v.1.30, http://www.mothur.org/) was used to calculate the Chao1 index to characterize species diversity, and the Shannon and Simpson diversity indices to characterize species diversity. Two-dimensional principal component analyses (PCA) plots and non-metric multidimensional scaling (NMDS) were used to assess the variation (β-diversity distance) between experimental groups.
Statistical analysis
All statistical analyses were performed using the ANOVA test. Results were represented as the mean ± SEM. Values were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.