Tong-Xie-Yao-Fang alleviates diarrhea-predominant irritable bowel syndrome by restoring intestinal barrier function via gut microbiota modulation and Wnt signaling pathway activation

Chemical Profiling of TXYF Decoction by High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS)For chemical characterization, 500 mg of lyophilized TXYF powder was accurately weighed and dissolved in 5 mL ultrapure water by vortexing for 5 minutes. Subsequently, 5 mL of LC-MS grade methanol was added, followed by vortex mixing for 1 minute and sonication for 60 minutes. The mixture was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22-μm membrane prior to analysis.HPLC-MS/MS analysis was performed on a Nexera LC-40 system (Shimadzu, Kyoto, Japan) equipped with a PDA detector and coupled to a Q Exactive Plus quadrupole-Orbitrap mass spectrometer (Thermo Scientific, USA) with a Heated Electrospray Ionization (HESI) source. Chromatographic separation was achieved using a Phenomenex Kinetex C18 column (2.6 μm, 2.1 × 100 mm). Data acquisition was controlled by Xcalibur 4.2 software in both positive and negative ionization modes.Abdominal Withdrawal Reflex (AWR) EvaluationVisceral hypersensitivity was quantified using the abdominal withdrawal reflex (AWR) score following colorectal distension [13]. A balloon-tipped catheter (originally for coronary angioplasty) connected to a sphygmomanometer was lubricated with glycerol and inserted into the colon until its tip was approximately 1 cm proximal to the anus. After acclimatization, the balloon was gradually inflated to graded pressures of 20, 40, 60, and 80 mmHg, with AWR scores recorded at each pressure according to established criteria. Loose Stool Rate DeterminationFollowing the treatment period, mice were individually housed in cages lined with sterile filter paper for a 4-hour observation period. Filter paper was replaced hourly to maintain cleanliness and ensure accurate counting. Fecal pellets were classified as diarrheal if they left visible wet stains on the filter paper. The loose stool rate was calculated as:Loose stool Rate = (Number of Unformed Pellets / Total Number of Fecal Pellets) × 100% Intestinal Permeability AssessmentIntestinal barrier function was evaluated using FITC-dextran 4000 (FD4) as described previously [14]. After a 24-hour fasting period, mice were orally administered FD4 solution (120 mg/mL) at a dose of 0.5 mL per 100 g body weight. Blood was collected from the portal vein after a standardized circulation period. Serum was obtained by centrifugation, and fluorescence intensity was measured using a microplate reader at excitation/emission wavelengths of 480/535 nm. Serum FD4 concentrations were determined using a concurrently generated standard curve. Sample Collection and ProcessingFecal samples were collected under sterile conditions from all groups following treatment completion and immediately stored at −80°C for subsequent metagenomic analysis. Metagenomic AnalysisMicrobial DNA was extracted from fecal samples using the OMEGA Stool DNA Kit. Following quality verification and quantification via Qubit fluorescence detection, DNA was randomly fragmented to construct sequencing libraries. Paired-end sequencing (PE150) was performed on an Illumina NovaSeq platform using a whole-genome shotgun (WGS) strategy, with raw data quality assessed using FastQC. Metagenomic sequences were assembled with MEGAHIT, followed by gene prediction and open reading frame (ORF) identification using MetaGeneMark. Non-redundant protein sequences were functionally annotated through alignment against standard protein databases. Alpha-diversity indices were compared across groups using one-way ANOVA with Tukey's HSD post hoc test (P < 0.05). Beta-diversity was assessed using Bray-Curtis distance matrices and statistically evaluated by PERMANOVA (Adonis), with visualization through principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). LEfSe analysis identified significantly differentially abundant taxonomic biomarkers. Functional profiling of KEGG orthologs utilized the ReporterScore algorithm with Wilcoxon testing for pairwise comparisons. Species-pathway correlations were examined using Spearman rank correlation and visualized as signed clustering heatmaps generated with OmicStudio. Histological EvaluationHematoxylin and Eosin (HE) StainingIleal tissues were fixed in 4% paraformaldehyde, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned. Following deparaffinization and hydration, sections were stained with hematoxylin and eosin. Villus height and crypt depth were measured using ImageJ software (National Institutes of Health, USA). Periodic Acid-Schiff (PAS) StainingIleal tissue segments processed similarly to HE samples were subjected to PAS staining to identify and quantify goblet cells. Morphometric analysis of goblet cell number, density, and morphology was performed using ImageJ software. Scanning Electron Microscopy (SEM)Ileal tissues were rinsed with cold PBS and fixed in pre-chilled 2.5% glutaraldehyde at 4°C for 2–4 hours. After rinsing, samples were post-fixed in 1% osmium tetroxide at 4°C for 1–2 hours, dehydrated through graded ethanol, substituted with isoamyl acetate, and critical-point dried. The dried specimens were mounted on stubs, sputter-coated with gold-palladium alloy, and examined under a scanning electron microscope. Statistical AnalysisStatistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). Data are expressed as mean ± standard deviation. Inter-group differences were assessed by one-way ANOVA followed by Tukey's post hoc test for homogeneous variances or Games-Howell test for heterogeneous variances. All tests were two-sided, with statistical significance defined as P < 0.05. Graphical representations were created using GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA).