Urolithin A-mediated augmentation of intestinal barrier function through elevated secretory mucin synthesis

MUC2 protein levels in the colonic mucosal epithelium were significantly higher in the Uro A group than that in the control group (control group; 100.0 ± 35.8, Uro A group; 142.0 ± 33.6, p = 0.041; Fig. 1d). In Nrf2-deficient mice, Uro A treatment did not increase MUC2 protein levels in the colonic epithelium (control group; 100.0 ± 48.0, Uro A group; 75.6 ± 36.8, p = 0.309, Wilcoxon rank sum test, Fig. 1e). Similarly, in mice treated intraperitoneally with an AhR antagonist, MUC2 protein levels did not increase in the colonic epithelium after treatment with Uro A (control group; 100.0 ± 20.3, Uro A group; 82.8 ± 26.1, p = 0.222, Fig. 1f).

Fluorescence immunostaining showed that the strength of MUC2 fluorescence (recognized as a reddish color) in LS174T cells after treatment with Uro A was significantly higher than that in the control group (control group; 93.9 ± 22.9, Uro A group; 131.9 ± 9.7, p = 0.030). In addition, MUC2 fluorescence expression did not increase after treatment with ML 385 (Nrf2 inhibitor, control group; 94.7 ± 9.0, Uro A group; 72.6 ± 19.2, p = 0.194) (Fig. 2c,d). Similarly, MUC2 fluorescence did not increase after treatment with CH223191↗ (AhR antagonist, control group; 88.3 ± 12.3, Uro A group; 86.7 ± 5.4, p = 1.000) (Fig. 2e,f).

Since previous reports have demonstrated that Uro A enhances tight junction¹³, we evaluated the mRNA expression of tight junction proteins. The mRNA expression of zo-1, zo-2, occludin, JAM, and claudin 1, 2, 3, 4, and 7 did not differ significantly between the Uro A and control groups (Fig. 3b).

After exposure to TNBS, mice exhibited severe colitis in the colon, as indicated by hyperemia, edema, wall hypertrophy, and ulcerative lesions, reflected in the macroscopic score. Uro A treatment reduced the macroscopic score after TNBS administration (p = 0.077) (Fig.a–c). S2

Six-week-old male WT mice (C57BL/6) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Similarly, six-week-old male Nrf2-deficient mice were purchased from the RIKEN Bioresource Center through the National BioResource Project (Ibaraki, Japan). The mice were caged individually in a room maintained at 18–24 °C with 40–70% relative humidity and a 12-h light/dark cycle. Mice had unrestricted access to food and potable water and were fed the rodent diet CE-2 (Nihon Clea, Tokyo, Japan) for 7 days during acclimatization.

All chemicals were prepared immediately before use. Uro A was generously provided by the Daicel Corporation (Osaka, Japan). The AhR antagonist CH-223191 and Nrf2 inhibitor ML385 were purchased from Selleck Biotech (Houston, TX, USA). All other chemicals were of the highest commercially available quality.

Male WT mice were divided into the control and Uro A-treated groups. Uro A, dissolved in carboxymethyl cellulose (CMC) at a dosage of 20 mg/kg or 100 mg/kg, was administered orally twice daily for 7 days as an exploratory experiment. In the control group, CMC was administered orally twice daily for 7 days. mRNA expression analysis of MUC2 in the colon epithelium indicated a gradual increase in MUC2 levels in the control, 20 mg/kg Uro A, and 100 mg/kg Uro A group (Fig. S1). To elucidate the mechanism by which Uro A produces mucus, we used the 100 mg/kg dose, which was more effective, although the previous report used a 20 mg/kg dose. We also included Nrf2-deficient (6 weeks old) and WT (C57BL/6) mice treated with an AhR antagonist (six mice per group). For AhR antagonism, CH-223191 was dissolved in corn oil, and 10 mg/kg was administered intraperitoneally 24 h before Uro A administration³⁹.

The collected colon tissues were fixed in Karnois solution for 3 h and immersed in ethanol. The anti-MUC2 antibody was used to stain the colon sections to highlight the MUC2 expression. Measurements of MUC2 thickness were conducted at nine distinct sites within each experimental group at 40× magnification. The computed averages of these measurements constituted the data used in this study.

Using the methodology described by Ohkawa et al.⁴⁰, MUC2 was quantified in the colonic mucosa as a marker for mucin production. After the experimental procedures, the colon mucosa (4 cm length of the distal colon) was scraped off using two glass slides, followed by homogenization in 1.5 mL of 10-mmol/L potassium phosphate buffer (pH 7.8) containing 30 mmol/L KCl using a Teflon Potter–Elvehjem homogenizer. The MUC2 concentration within the tissue homogenates was measured using the SEA705Mu Mucin enzyme-linked immunosorbent assay (ELISA) kit model (Cloud-Clone Corp. Wuhan, China), in strict accordance with the manufacturer’s protocol. For MUC2 concentrations, each control group was settled as 100.

FITC-dextran (Krackeler Scientific Inc., Albany, NY) was used to investigate the alterations in colon permeability. A dose of 4 kDa FITC-dextran (10 mg/0.25 mL/mouse) was orally administered, and plasma samples were collected from the portal vein 3 h post-administration^41,42. FITC-dextran is a valuable tool for assessing intestinal permeability, especially because it remains in the colon until excreted, with 4 kDa FITC-dextran being the optimal molecular weight^43,44.

Feces collected from the mice were stored in a freezer at − 80 °C until DNA extraction. Genomic DNA was extracted using a NucleoSpin Microbial DNA Kit (Macherey–Nagel, Düren, Germany). Nine variable regions (V1–V9) in 16S rRNA provide the most useful information for phylogenetic and taxonomic studies. A two-step polymerase chain reaction (PCR) was performed on the purified DNA samples to obtain sequence libraries. The first PCR was performed to amplify and use a 16S (V3–V4) metagenomic library construction kit for NGS (Takara Bio Inc., Kusatsu, Japan) with the primer pairs 341F (5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3ʹ) and 806R reverse primer (5ʹ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3ʹ) that corresponded to the V3–V4 region of the 16S rRNA gene.

The second PCR was performed to add the index sequences for the Illumina sequencer with a barcode sequence using the Nextera XT index kit (Illumina, San Diego, CA, USA). The prepared libraries were subjected to 250 paired-end base sequencing using the MiSeq Reagent v3 kit and MiSeq (Illumina) at the Biomedical Center of Takara Bio.

Quantitative Insights into Microbial Ecology 2 was used to analyze the sequence data. The DADA2 model was used to denoise the sequence reads, and the amplicon sequence variant (ASV) and representative sequences were determined. The ASVs were taxonomically assigned using a sklearn classifier against the GreenGenes database (13_8). Principal coordinate analysis was used to compare beta-diversity based on UniFrac distances, and permutational analysis of variance was used for statistical analysis of beta-diversity. From the obtained gut microbiota data, linear discriminant and effect size analyses were performed to identify the alternation in the abundance of the gut microbiota in mice after 1 week of Uro A administration.

Six-week-old male WT mice (C57BL/6) were used for the study. An experimental acute colitis model was induced using 2.5% DSS (molecular weight: 36,000–50,000 Da; lot no. M8667; MP Biomedicals, Santa Ana, CA, USA) in the drinking water for 7 days. The mice were euthanized under anesthesia precisely 7 days after the initiation of DSS treatment.

We evaluated colon length and the DAI, which was calculated by scoring changes in stool consistency, occult blood positivity, gross bleeding, and body weight, as previously described⁴⁵. We used three grades of stool consistency (0, normal; 2, loose; and 4, diarrhea), three grades of occult blood (0, negative; 2, occult blood-positive; and 4, gross bleeding), and five grades of weight loss (0, no loss or weight gain; 1, 1–5% loss; 2, 5–10% loss; 3, 10–20% loss; and 4, > 20% loss). After evaluating the DAI, the mice were sacrificed, the entire colon was resected from the cecum to the anus, and colon length was measured as an indirect marker of intestinal inflammation. Distal colon specimens were preserved in 10% neutral buffered formalin for histological evaluation. Post-fixation, the specimens were embedded in paraffin, split into 7-µm sections, and subjected to hematoxylin and eosin staining.

Colitis was induced in mice that were minimally anesthetized with ketamine/xylazine via a catheter by intrarectal administration of 200 mg/kg TNBS (Sigma–Aldrich Japan, Tokyo, Japan) dissolved in 30% ethanol^46,47.

Mice in the control group were administered 30% ethanol. After 3 days post-TNBS administration, the mice were euthanized, and their colons were extracted for examination. The inflicted colonic damage was evaluated and ranked based on predetermined criteria. Macroscopic grading included visible impairments, serosal adhesions, diarrhea, strictures, and increased bowel wall thickness. All grading was executed by a singular individual under blinded conditions, thereby preventing observer bias.

LS174T is a human colon adenocarcinoma cell line registered under the CL‑188TM mark at the American Type Culture Collection (Manassas, VA, USA). This cell line, which demonstrates traits of mucin-secreting intestinal epithelial cells, has been extensively used as a representative intestinal goblet cell line. Cultivation of LS174T cells spanned 1 week; it was performed using Dulbecco’s Modified Eagle Medium (DMEM) fortified with 2 mM l-glutamine, 10% heat-inactivated fetal bovine serum (FBS), and 100 U/mL penicillin. The incubation conditions comprised a temperature of 37 °C within a humidified atmosphere containing 5% carbon dioxide and 95% air. LS174T cells, at a concentration of 2.5 × 10⁵ cells/mL, were distributed in 6‑well plates in preparation for the subsequent ELISA assay. CH-223191, an AhR antagonist, was dissolved in 100% dimethyl sulfoxide (DMSO), and a 10-µM concentration was administered. Similarly, ML385, functioning as an Nrf2 inhibitor, was prepared by dissolving it in 100% DMSO to create a stock solution and subsequently diluted to a 10-µM concentration before application⁴⁸.

LS174T cells were cultured in a 24-well-plate or μ-dish (35-mm) imaging dishes (ibidi GmbH, Martinsried, Germany) until the point of complete confluence. The cells were treated with DMEM without FBS for 12 h before Uro A treatment. In this experiment, Uro A was dissolved in DMSO at a concentration of 10 µM. LS174 cells were cultured, followed by exposure to 10 µM Uro A for 3 h to quantify the concentration of secreted MUC2. The amount of MUC2 protein present in the cell supernatant was determined using a Mucin ELISA kit, specifically the SEA705Hu model (Cloud-Clone Corp. Wuhan, China), according to the procedural guidelines provided by the manufacturer.

LS174T cellular specimens were cultivated on 35-mm μ-dishes intended (ibidi GmbH, Martinsried, Germany) and subsequently exposed to a concentration of 10 µM Uro A for 24 h. Following this, the cellular specimens were stabilized using 4% paraformaldehyde dissolved in PBS for 10 min, permeated with a PBS solution incorporating 0.1% Triton X-100 for 5 min, and subjected to a 24-h incubation period at a temperature of 37 ℃ with a primary antibody, specifically ab97386, targeting MUC2. In the subsequent stage, the cells were incubated with goat anti-rabbit Alexa 594, rhodamine-phalloidin, and Hoechst 33352 secondary antibodies, employed as staining agents for MUC2 and F-actin. Hoechst 33352 was used to stain nuclear chromatin. In the final step, cellular specimens were meticulously examined using the all-in-one fluorescence BZ-X810 laser scanning microscope (Keyence, Milton Keynes, UK). Fluorescence intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

All experiments were performed in accordance with the ARRIVE guidelines 2.0 and the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th edition, 2011) and approved by the Institutional Ethical Committee for Animal Experiments of the Kyoto Prefectural University of Medicine (Kyoto, Japan) under Assurance Number M 2020-113.

All analyses were performed using the JMP PRO version 14.0.0 (SAS Institute Japan Ltd.). The trend test was performed using linear contrast. The evaluation of average tendencies, segmented based on customarily distributed continuous parameters, was performed using an analysis of variance. In normally distributed data, continuous variables are represented as means with an additional or subtractive standard deviation. For non-normally distributed data, the data were expressed as the median and the interquartile range (IQR) (25%, 75%).

Supplementary Legends. Supplementary Figure S1. Supplementary Figure S2.