Hyperoside was purchased from MUST Biotechnology Co., Ltd. (Chengdu, China). DSS (MW 36,000~50,000, 160110, MP) was obtained from MP Biomedicals (CA, USA). The following antibodies were used in this study: anti-ZO-1 antibody (AF5145, Affinity), anti-mucin-2 antibody (GB11344, Serbicebio), anti-BDNF antibody (GB11559, Serbicebio), anti-GFAP antibody (GB11096, Serbicebio), anti-Iba-1 antibody (GB154490, Serbicebio), NF-κB p65 (Proteintech, 10745-1-AP), Phospho-NF-κB p65 (Affinity, AF2006), AKT (Proteintech, 10176-2-AP), Phospho-AKT (Proteintech, 28731-1-AP), p38MAPK (Proteintech, 51115-1-AP), Phospho-p38MAPK (Affinity, AF4001). Antibiotics for microbiota depletion, including ampicillin (A102048), metronidazole (B300250), vancomycin (V301569),and neomycin (N412785) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China).

Male C57BL/6 mice (6–8 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. and maintained under specific pathogen-free (SPF) conditions with a controlled environment (12 h light/dark cycle, 25 ± 2°C, 50–55% humidity). All experimental procedures were approved by the Ethical Review Committee of The First Hospital Affiliated with Shandong First Medical University & Shandong provincial Qianfoshan Hospital (application number: 2025070901). Following a 1-week acclimatization period, mice were randomly divided into five experimental groups (n = 10 per group): Control group: Received normal drinking water and equivalent volume of vehicle by daily oral gavage. DSS group: Administered 3% DSS in drinking water ad libitum for 7 days with vehicle gavage. DSS + HYP 10 mg/kg group: Treated with 3% DSS plus low-dose HYP (10 mg/kg/day) by oral gavage. DSS + HYP 30 mg/kg group: Treated with 3% DSS plus high-dose HYP (30 mg/kg/day) by oral gavage. DSS + 5-aminosalicylic acid (5-ASA) group: Received 3% DSS plus 5-ASA (100 mg/kg/day) by oral gavage. The dosages of HYP used in this study were determined based on previous research (Cheng et al., 2021).

In a separate experiment to evaluate the microbiota-dependent therapeutic effects of HYP, mice were randomly assigned to three experimental groups (n = 5 per group) with the following treatments: ABX group: Received daily oral gavage of a broad-spectrum antibiotic cocktail (1 mg/mL ampicillin, 1 mg/mL metronidazole, 0.5 mg/mL vancomycin, 1 mg/mL neomycin) for 5 consecutive days to deplete gut microbiota, followed by regular drinking water for 7 days. ABX + DSS group: Underwent identical antibiotic pretreatment as the ABX group, followed by 3% DSS administration in drinking water for 7 days to induce colitis under microbiota-depleted conditions. ABX + DSS + HYP group: Received the same antibiotic and DSS regimen as the ABX + DSS group, with concurrent daily oral administration of HYP (30 mg/kg) for 7 days during the DSS exposure period.

Distal colon specimens were fixed in 4% paraformaldehyde for 24 h at 4°C, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin.

Serial 4-μm sections were cut, deparaffinized in xylene, and rehydrated in descending ethanol concentrations. Sections were stained with freshly filtered hematoxylin staining solution (3–5 min) to visualize nuclei, counterstained with eosin staining to highlight cytoplasmic structures, dehydrated through graded ethanol and xylene, and coverslipped with neutral balsam. Histopathological evaluation was performed according to the scoring system described previously (Chen et al., 2025).

Total RNA was extracted from colon tissues using the FastPure RNA Kit III. Following genomic DNA removal, cDNA was synthesized from 1 μg RNA using the PrimeScript™ RT Reagent Kit (Takara Bio). qRT-PCR assays were performed in triplicate using SYBR Premix Ex Taq II on a QuantStudio 7 Flex system. The thermal cycling conditions were as previously described (Chen et al., 2019). Relative gene expression was calculated via the 2^-ΔΔCT method normalized to Gapdh. Primer sequences are listed in Table 1.

Paraffin-embedded tissue sections were deparaffinized and rehydrated. For immunofluorescence, sections were blocked with BSA and incubated overnight at 4°C with primary antibodies, followed by appropriate secondary antibodies and DAPI counterstaining. For immunohistochemistry, antigen retrieval was performed using EDTA buffer, followed by incubation with primary and secondary antibodies. Signal was developed using DAB substrate, and sections were counterstained with hematoxylin. Images were captured using a fluorescence or bright-field microscope, respectively.

Levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in serum were quantified with specific ELISA kits (Cusabio, Wuhan, China). The assay was performed following the manufacturer’s protocols. The absorbance of each sample was measured with a microplate reader, and the corresponding cytokine concentrations were determined by interpolation from a standard curve.

The open field test was conducted to evaluate anxiety-related behaviors in mice using an established protocol with minor modifications. The testing apparatus consisted of a black polyvinyl chloride square arena (50 × 50 × 40 cm) with white flooring. The testing room was maintained under dim illumination with minimal environmental noise. Each mouse was gently placed in a corner of the arena and allowed to freely explore for 5 minutes (300 seconds). Between trials, the arena was thoroughly cleaned with 75% ethanol to eliminate odor cues. Total distance traveled (mm), average movement velocity (mm/s), time spent in central zone (% of total trial duration) and time spent in peripheral zone (% of total trial duration) were calculated.

The elevated plus maze test was performed to evaluate anxiety-like behaviors following established protocols with modifications. The apparatus consisted of four perpendicular arms (30 cm long × 5 cm wide) arranged in a “+” configuration elevated 50 cm above the floor. Two opposing arms were enclosed by 15-cm high opaque walls (closed arms), while the other two arms had no walls (open arms). All arms extended from a central square platform (5 × 5 cm). Prior to testing, mice were acclimated to the testing room for 30 minutes under dim light. Each mouse was gently placed in the central platform facing an open arm and allowed to freely explore the maze for 200 seconds. Behavioral sessions were recorded using a video tracking system. Total distance traveled (mm) and average movement velocity (mm/s) was calculated.

Colon tissue proteins were extracted using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (all from Beyotime). Protein concentrations were determined with a BCA assay kit (Proteintech). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence detection system (Tanon).

Fecal metabolomic profiling was performed using the Q300 Kit (Metabo-Profile, Shanghai, China). Briefly, fecal samples were homogenized with zirconium oxide beads in a mixture of deionized water and methanol containing internal standards. After centrifugation, the supernatant was collected and derivatized at 30 °C for 60 min using a Biomek 4000 workstation (Beckman Coulter, USA). The derivatized samples were reconstituted in 50% methanol and subjected to LC-MS analysis.

Analysis was conducted on an ACQUITY UPLC-Xevo TQ-S system (Waters Corp., USA). Separation was achieved on a BEH C18 column (2.1 × 100 mm, 1.7 μm) maintained at 40°C, with a mobile phase consisting of (A) 0.1% formic acid in water and (B) acetonitrile:isopropanol (70:30). The gradient elution program was as follows: 0–1 min (5% B), 1–11 min (5–78% B), 11–13.5 min (78–95% B), 13.5–14 min (95–100% B), 14–16 min (100% B), 16–16.1 min (100-5% B), 16.1–18 min (5% B), flow rate: 0.40 mL min-1, and injection vol.: 5.0 µL. Mass spectrometry was performed in both ESI positive and negative modes with capillary voltages of 1.5 kV and 2.0 kV, respectively. The source and desolvation temperatures were set at 150°C and 550°C, respectively.

Raw data were processed using the iMAP platform (v1.0, Metabo-Profile). Multivariate analyses, including principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), were performed. Differentially expressed metabolites (DEMs) were identified based on a variable importance in projection (VIP) score of ≥ 1 from the OPLS-DA model and a p-value < 0.05 from univariate analysis.

The common targets were submitted to the Metascape platform for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The analysis parameters were set as follows: H. sapiens as the species, a p-value cutoff of 0.01, a minimum overlap of 3, and a minimum enrichment of 1.5. The top 20 significantly enriched pathways, ranked by p-value, were selected for visualization. Visualize the KEGG results using the online data analysis and visualization platform Bioinformatics (http://www.bioinformatics.com.cn). Map the top 10 pathways by P-value to the potential functional targets of HYP treatment for UC in a cytoscape-style pathway mapping.

The three-dimensional crystal structures of the core target proteins were obtained from the RCSB-PDB database (https://www.rcsb.org/). All protein macromolecular structures were processed using PyMOL v2.5 (open-source version) to remove original ligands, water molecules, and unnecessary ions, retaining only the polypeptide chains necessary for docking. Subsequent preparation, including the addition of hydrogen atoms and assignment of charges, was performed using AutoDockTools to generate the final protein structures in PDBQT format.

The 3D chemical structure of Hyperoside was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and its SDF format file was downloaded (Table 2). Open Babel (v3.1.1) was used for file format conversion and energy minimization. The prepared ligand structure was then processed with AutoDockTools to define rotatable bonds and output in PDBQT format.

Molecular docking was performed using AutoDock Vina (v1.2.5). The docking grid was configured to encompass the entire known active site or potential binding pocket of each protein. For each target, nine docking poses were generated, and the conformation with the most favorable (lowest) binding energy was selected as the optimal binding mode.

The optimal docking complexes were visualized using PyMOL to analyze the binding conformation and intermolecular interactions, such as hydrogen bonds and hydrophobic contacts. Furthermore, the PoseView server (https://proteins.plus/) was employed to generate a two-dimensional interaction diagram of Hyperoside bound to the protein, enabling analysis of the specific interaction types between key residues and the ligand.

All quantitative data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.0. Differences between two groups were assessed using an unpaired, two-tailed Student’s t-test. For comparisons among more than two groups, one-way analysis of variance (ANOVA) was employed. A p-value of less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

To evaluate the therapeutic effects of HYP on UC, mice first underwent a 7-day acclimatization period. Subsequently, colitis was induced by providing them with 3% DSS in drinking water for 7 days. During the DSS administration period, mice were concurrently treated with either HYP or 5-ASA. The experimental timeline is illustrated in Figure 1A. Mice in the DSS group exhibited significant body weight loss compared to the Ctrl group. In contrast, both HYP and 5-ASA treatments markedly attenuated this DSS-induced weight reduction (Figure 1B). Furthermore, colon lengths were significantly shorter in the DSS group than in the control group, whereas treatment with a high dose of HYP (30 mg/kg) or 5-ASA substantially reversed this shortening (Figures 1C, D). Histological analysis revealed severe mucosal damage in the DSS group, characterized by crypt structure destruction, loss of goblet cells, and substantial inflammatory cell infiltration. These pathological changes were notably alleviated by HYP and 5-ASA treatment, accompanied by significantly lower histopathological scores in the intervention groups (Figures 1E, F). Collectively, these findings demonstrate that HYP administration ameliorates DSS-induced UC in mice.

To investigate the impact of HYP on UC-associated anxiety and depressive-like behaviors, we performed a series of behavioral tests. In the OFT (Figure 2A), DSS-treated mice exhibited a significant reduction in total moving distance (Figure 2B), average velocity (Figure 2C), and time spent in the central zone (Figure 2D) compared to the Ctrl group. These behavioral deficits were markedly reversed by high-dose HYP treatment. Consistent with these findings, DSS administration increased the time spent in the marginal regions of the arena, a trend that was also counteracted by HYP intervention (Figure 2E). The EPM test yielded complementary results (Figure 2F). Mice in the DSS group showed a significant decrease in both the total distance traveled (Figure 2G) and the average movement velocity (Figure 2H) compared to controls. High-dose HYP treatment notably restored these activity parameters. Collectively, these behavioral findings demonstrate that HYP treatment effectively attenuates anxiety-like behaviors in a murine model of DSS-induced colitis.

To assess the impact of HYP on systemic inflammation, we measured key inflammatory cytokines. DSS administration significantly elevated the levels of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β in mouse colon tissues and serum compared with the Ctrl group. Treatment with HYP, however, effectively suppressed these DSS-induced increases (Figures 3A–C, E-G). We further investigated whether HYP could protect the intestinal barrier. Immunofluorescence analysis revealed that DSS challenge markedly reduced the protein expression of Mucin-2, a critical component of the mucosal layer, compared to the control group. In contrast, HYP intervention significantly restored Mucin-2 expression (Figures 3D, I). Similarly, the expression of the tight junction protein ZO-1 was profoundly decreased by DSS but was notably upregulated after HYP treatment (Figures 3H, J). Taken together, these findings demonstrate that HYP ameliorates intestinal inflammation and promotes restoration of barrier integrity in mice with DSS-induced colitis.

To determine whether HYP modulates the brain response to peripheral inflammation, we first examined brain histopathology. Hematoxylin and eosin (H&E) staining revealed no overt morphological changes in the hippocampal region across all groups (Supplementary Figure 1A). Given the established roles of neurotrophic deficits and glial cell activation in neuroinflammation and anxiety-like behavior, we next assessed the expression of key molecular markers by immunohistochemistry. We analyzed brain-derived neurotrophic factor (BDNF) and the microglial/astrocyte activation markers Iba1 and GFAP, respectively. As shown in Figure 4, DSS administration significantly downregulated BDNF protein levels in multiple brain regions, including the cortex (Figure 4A), dentate gyrus (DG) (Figure 4B), CA1 (Figure 4C) and CA3 (Figure 4D) hippocampal areas, compared to the Ctrl group. HYP treatment effectively reversed this DSS-induced reduction in BDNF (Figures 4F-I). Conversely, DSS challenge markedly upregulated the expression of Iba1, a marker of microglial activation. This increase was significantly suppressed by HYP intervention (Figures 4E, J). In contrast, the expression of GFAP, an astrocyte marker, showed no significant alterations among the groups (Supplementary Figures 1B, C). These results indicate that HYP mitigates DSS-induced neuroinflammation, likely through the restoration of BDNF expression and suppression of microglial activation.

To assess the impact of HYP on the gut microbial community, we first analyzed both alpha and beta diversity. Significant differences were observed in alpha diversity indices, including Chao1 (Figure 5A), Shannon (Figure 5B), and Simpson (Figure 5C), among the Control, DSS, and HYP-treated groups. Principal component analysis (PCA) further revealed distinct clustering of microbial communities, indicating differences in overall composition (beta diversity) across groups (Figure 5D). To elucidate the specific taxonomic shifts underlying these changes, we profiled the microbiota at the phylum and species levels. At the phylum level, HYP treatment reversed the DSS-induced microbial imbalance, characterized by a lower relative abundance of Proteobacteria and a higher abundance of Firmicutes, similar to the profile observed in the control group (Figure 5E). Further analysis at the species level showed that HYP intervention significantly increased the abundance of Enterobacter ludwigii (Figure 5G) while reducing the levels of Enterobacter hormaechei (Figure 5H), Escherichia coli (Figure 5I), and Acinetobacter baumannii (Figure 5J). LEfSe analysis (LDA score > 3.0) identified s:Enterobacter ludwigii as a key biomarker enriched in the HYP-treated group (Figure 5K), suggesting its potential involvement in the therapeutic mechanism of HYP against UC. Notably, Enterobacter ludwigii has been previously reported to mitigate colitis, restore intestinal barrier integrity, and promote gut health (Li et al., 2022). Collectively, these findings indicate that HYP administration effectively alleviates DSS-induced dysbiosis and restructures the gut microbiota composition.

To investigate the metabolic alterations underlying the therapeutic effects of HYP, we conducted a comprehensive quantitative metabolomic analysis using the Q300™ platform. Orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed clear metabolic separations between the Ctrl and DSS groups (Figure 6A), as well as between the DSS and HYP-treated groups (Figure 6B), indicating a significant shift in the global metabolite profile following HYP intervention. Subsequent KEGG pathway enrichment analysis of the differentially abundant metabolites showed that HYP treatment significantly enriched several metabolic pathways. Most notably, the arginine biosynthesis pathway was prominently enriched, along with pathways for alanine, aspartate, and glutamate metabolism, and glyoxylate and dicarboxylate metabolism (Figures 6C, D). Consistent with the pathway analysis, HYP intervention significantly increased the relative abundances of key metabolites within the arginine biosynthesis pathway, including arginine (Figure 6E), glutamic acid (Figure 6F), glutamine (Figure 6G), and aspartic acid (Figure 6H). Taken together, these findings demonstrate that HYP remodels the host-microbiota metabolome and specifically enhances arginine metabolism, which may contribute to its protective effects against UC.

To systematically investigate the underlying mechanisms of HYP against ulcerative colitis (UC), we performed a network pharmacology analysis. A total of 661 HYP-related targets and 2,852 UC-related targets were identified, with 309 overlapping targets considered as potential therapeutic targets of HYP for UC (Figure 7A). A protein-protein interaction (PPI) network was constructed using these common targets (Figure 7B). Subsequent KEGG pathway enrichment analysis revealed that significantly enriched pathways included the PI3K-Akt, MAPK, and NF-κB signaling pathways, all of which have been previously implicated in UC pathogenesis (Figure 7C). The top 20 hub genes were identified through topological analysis based on degree values (Figure 7D). From these hub genes, three key candidates (MAPK3, AKT1, and NFKB1) were selected for subsequent molecular docking and experimental validation.

Molecular docking was performed to evaluate the binding affinity between HYP and these key proteins. MAPK3 showed the strongest binding affinity with HYP (−8.745 kcal·mol^-1) (Figure 7E), followed by AKT1 (−8.23 kcal·mol^-1) (Figure 7F) and NFκB1 (−7.31 kcal·mol^-1) (Figure 7G). The information of HYP is shown in Table 2. The optimal docking pose for each protein is summarized in Table 3, with all nine docking poses provided in Supplementary Table 1. To further validate these findings, Western blot analysis was conducted to assess the expression of pathway-related proteins (Figure 7I). These integrated results suggest that the therapeutic effects of HYP on UC may be mediated through the modulation of multiple signaling pathways, particularly the MAPK, PI3K-Akt, and NF-κB pathways.

To determine whether the intestinal microbiota is required for the therapeutic effects of HYP, we employed an antibiotic depletion strategy (Figure 8A). In microbiota-depleted mice, HYP treatment failed to ameliorate DSS-induced body weight loss (Figure 8B) or colon shortening (Figure 8C). Consistent with this, histopathological analysis showed that HYP no longer mitigated DSS-induced crypt damage and inflammatory infiltration after antibiotic treatment (Figures 8D, E). We further assessed whether the behavioral benefits of HYP were also microbiota-dependent. In the OFT, HYP did not improve the reduced moving distance or average velocity in microbiota-depleted mice (Figures 8F, G). Similarly, in the EPM test, the restorative effects of HYP on total distance traveled and movement velocity were abolished following antibiotic treatment (Figures 8H, I). Collectively, these findings demonstrate that the intestinal microbiota is essential for the efficacy of HYP in alleviating both colitic pathology and associated anxiety-like behaviors.